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How Enzymes Handle the Energy Derived from the Cleavage of High-energy Phosphate Compounds

Open AccessPublished:March 16, 2012DOI:https://doi.org/10.1074/jbc.X112.363200

      Introduction

      I was accepted at the Federal University of Rio de Janeiro Medical School when I was seventeen years old. At that time, I was absolutely sure I was going to be a surgeon. One year later, I received an offer for a fellowship to work in a research laboratory. This originally had not been part of my future professional plan, but I was desperately in need of a source of income, so I joined the research laboratory at the age of eighteen. During the medical school course in Rio de Janeiro, I lived the hormonal tempest typical of youth, and my certainties were shaken badly. I soon discovered that I did not have much of a future in either surgery or clinical practice, but my interest in science increased. I received my M.D. degree in 1961, when I was twenty-three years old, and it was time to decide what to do with my life. With a significant degree of uncertainty, I opted for research. Fortunately, I had excellent supervisors who smoothed out my rite of passage into science.
      As a medical student in Rio de Janeiro, I worked with Walter Oswaldo Cruz (Fig. 1) in his research laboratory, and shortly after receiving my M.D. degree, I went to the National Institutes of Health (NIH) in Bethesda, where I had the good fortune to work with Herbert and Celia Tabor (Fig. 2) for eighteen months. When I returned to Rio de Janeiro, I was sure that my vocation was in science. I was not yet what one could call a scientist, but I had the basic skills to begin a career.
      Figure thumbnail gr1
      FIGURE 1Walter Oswaldo Cruz, my tutor during my initial period as a medical student.
      Figure thumbnail gr2
      FIGURE 2Herbert and Celia Tabor, my tutors during my postdoctoral training at the NIH in Bethesda. They taught me what biochemistry is.

      About Science and Scientists

      My major interest throughout the years has been the mechanism by which enzymes interconvert different forms of energy. This involves the concept of high- and low-energy phosphate compounds and the way some proteins regulate how much of the energy available can be used to perform work and how much is converted into heat. When I first started to work in research, I thought of myself as a physiologist with some training in biochemistry. If someone had told me at that time that he worked in the subjects I have just described, I initially would have had difficulty understanding what he was saying. I would have believed that his work required solid physicochemical training, which I did not have. I would also have thought that the application of this sort of research would be related only remotely to possible medical applications, to which I always hoped to contribute. This juvenile reasoning proved to be completely wrong.
      Like any dedicated researcher, I have received some awards for my work, but the sources of these awards often have been unexpected. For example, once I was made an honorary member of the Brazilian Academy of Medicine, and I thought, “I am not ignored by the medical doctors.” At home, when I announced that I was still an M.D. after all, my second wife (Fig. 3), my two sons, and my two daughters, who all practice in various medical fields, burst into laughter. I felt their reaction was cruel, but deep down, I had to agree with them because I was a researcher. I then received a nice international award for my work in chemistry, and I was later quoted in several articles published in journals of physics. No one at home laughed at me when I told them of these accomplishments; on the contrary, they said, “Bravo, Papa!”
      Figure thumbnail gr3
      FIGURE 3My second wife, Vivian Rumjanek, and me. She is an immunologist. My first wife, Regina, was a geologist. We had two daughters and two sons, but unfortunately, she died at an early age.
      As time passed and maturity quieted my emotions, I realized that the work itself is what is important, not whether it is categorized as medicine, chemistry, or whatever. When working, there are magic moments in which one suddenly unravels a new finding and has the sensation of understanding a small facet of nature. The magical moment has a short duration, and the new finding is cleared up and substantiated with controls. Then comes the tedious part: writing the paper, submitting it for publication, waiting to know if it has been accepted, and, at times, enduring the rudeness of a bad-tempered referee until finally it is published. At this stage, the magic has faded away, but one goes on struggling to discover something new in the hopes of once more feeling the sensation of a new discovery, no matter the field or how significant it may be. In this search, the main tool frequently is intuition (
      • de Meis L.
      • Longo P.H.
      • Falcáo E.B.M.
      The learning process in science: a study among Brazilian biochemists.
      ). I think scientists are addicts of those glorious moments that follow discovery, and it is with the hope of feeling again the thrill of these moments that they endure long hours of work investigating whatever subject intuition suggests.
      I have three major interests related to my profession: research, education, and social studies in science (
      • de Meis L.
      • Longo P.H.
      • Falcáo E.B.M.
      The learning process in science: a study among Brazilian biochemists.
      ,
      • de Meis L.
      ,
      • de Meis L.
      • Leta J.
      Modern science and the explosion of new knowledge.
      ). I worked most of my life as a university professor and used to select my undergraduate students during my courses on medicine and biology. As my interest in education increased, I started to organize short experimental courses in different Brazilian universities for young students at what would be the equivalent of the high school level in the United States. These young people live in the lower income neighborhoods of Rio de Janeiro periphery and cannot afford the proper education necessary to attend university. The experimental courses are totally free, and the best students are invited to work in the laboratory under the supervision of M.Sc. or Ph.D. students and eventually enter public universities, which, in Brazil, are free of charge. The students receive a small stipend and all the educational materials they need. Many of these students have graduated and gone on to earn doctorate degrees and have obtained good positions. The system has spread throughout the whole country, and the government is now allocating funds for this activity in the form of science grants. The writer Malcolm Bradbury, in a romance about a professor of history, wrote that although he grew older over the years, his students were always eighteen years old. I have found this to be true, but in my case, I would expand this from fifteen to twenty-five years old. This experience with young people was of great help in educating my own four children and led me to do active research in education and science sociology. In this article, I will refer only to my work in bioenergetics and leave the education affair for another opportunity.

      From Italy to Brazil

      I was born to an Italian musician's family. My grandfather was a member of the Scala in Milan and the San Martino Theater in Naples. My father played the cello (Fig. 4), and he did well until fascism rose to power in Italy. Because of his antifascist attitudes, he first had problems getting a job and later had to hide from the regime. After the war, the economic situation in Italy was chaotic, and my father decided to emigrate to Brazil and become a member of the Brazilian Symphonic Orchestra (Fig. 4). When we left Italy, I was nine years old, and my father assured us that the move would be for only a short period until he became rich, and we could come back to a free Italy. He never got rich, nor did we ever return to Italy.
      Figure thumbnail gr4
      FIGURE 4Ezio de Meis, my father, who taught me to pursue my dreams no matter what.

      As I Became Addicted to Science

      I entered into a research laboratory for the first time in 1957, when I was an eighteen-year-old medical student. I worked under the supervision of Dr. Walter Oswaldo Cruz (Fig. 1) at the Institute that carried his family name (from his father). It was not easy to get a position in Dr. Oswaldo Cruz's laboratory. In addition to being an excellent scientist, he paid a hefty fellowship, the equivalent at present of 300 dollars per month. For someone from a modest family, this was a fortune. Therefore, I applied for a position as soon as one became available, even though, at the time, I envisioned a future as a surgeon, not a researcher.
      Dr. Oswaldo Cruz had a peculiar way of selecting his students. The first step of the selection took place at his home. When I arrived, the living room was crowded with candidates, most of whom were far more advanced in the medical course than I was. I feared that I did not stand a chance in comparison, but fortunately, I decided to stay and face the oncoming massacre. When I was finally called for the interview, to my surprise, I found out that Dr. Oswaldo Cruz did not care about my performance at the university or if I knew anything about hematology (his field), and he cared even less if I had any laboratory experience. After a very brief, casual conversation, he showed me several cartoons selected from the American magazine The New Yorker and asked me to identify the humorous point of the cartoon. After the interview, Dr. Oswaldo Cruz informed me that those selected would be called for a second interview. Many did not make it past the first step, but to my surprise, I was called for the second test in his laboratory. Here, he showed us complex pieces of equipment and asked us what they were used for. When I answered that I had no idea, he said, “Good. Now make a guess.” A week later I was informed that I had been selected and that I should go to his laboratory the following week. I was an exhilarated new apprentice. At first, I felt like a millionaire as I imagined the 300 dollars I would receive each month. Then, as I cooled down, I had the feeling that, in Dr. Oswaldo Cruz's view, the main ingredients for science were a good sense of humor and a lot of intuition. As the years passed, I found out that Dr. Oswaldo Cruz's recipe was a good one.
      I worked in Dr. Oswaldo Cruz's laboratory for six years. He used to design the experiments, and we, the students, performed them. Of course he accepted suggestions and encouraged us to test them, and while there, I learned what it meant to have a good sense of humor in science. When one of my suggestions was accepted, a rare occurrence, I worked crazily, skipping weekends, classes at the university, and encounters with my girlfriend. After a certain amount of time, I usually had to give up because my brilliant idea turned out to be not so brilliant. This was always excruciating and a terrible blow to my ego; I was not as brilliant as I wished to be. So I would go to Dr. Oswaldo Cruz and ask if he had any experiments that needed to be done in the main flux of the laboratory. He would smile (a cruel smile, to my understanding) and ask, “Why, your good suggestion did not work?” With a grotesque attempt at making a humorous face, I would answer, “Nature does not seem to appreciate my ideas.” This was stated causally, but inside, I wanted to kill, kick, and do all sorts of malignant acts. Fortunately, this frustration did not persist for a long period: I was soon distracted by the need to catch up on the lessons missed at the university and search for a new girlfriend because the previous one thought I had intentionally ignored her for weeks. I repeated this masochist ritual several times over the years, and a few of my suggestions actually worked out. Years later, during a friendly conversation, Dr. Oswaldo Cruz told me that he used this strategy to see if a trainee was able to face frustration without coming up with false interpretations or, even worse, “adapting” results. To my astonishment, Dr. Oswaldo Cruz also informed me that he also frequently suffered frustrations with ideas that he initially thought to be excellent.
      At the end of my medical course, I was a coauthor of several of Dr. Oswaldo Cruz papers that had been published in well known international journals and had authored a couple of small papers of my own. Dr. Oswaldo Cruz felt that I had a reasonable knowledge of physiology and insisted that I should work in the United States for one or two years to be initiated into the field of biochemistry. He helped me in all possible ways to get a fellowship. Thus, I had the good fortune to end up in the laboratory of Drs. Herbert and Celia Tabor (Fig. 2) at the NIH. The Tabors taught me many things, starting with discipline. For a young man raised in a tropical country, waking up at 6:45 a.m. during the winter when it was dark outside and snow was falling was extremely difficult. The working hours at the NIH were 8:30 a.m. to 5 p.m., but I used to come in between 9 and 10 a.m. and work until later in the evening both to compensate for the late arrival and because I enjoyed the work at the laboratory. One day, in a kind but firm voice, Herbert informed me that I had to start at 8:30 a.m. no matter how cold it was outside and then should feel free to work after 5 p.m. if this suited me. In the same kind tone of voice, Herbert told me that I had to attend the journal club meetings they conducted in Building 10. The journal club had been organized by Arthur Kornberg and was attended by distinguished scientists, including Bernard Horecker, Leon Heppel, and Maxine Singer. At the beginning, I could grasp very little of what was being presented, but in a few months, I could understand all of it.
      Usually, after the journal club meetings, I would have a generous lunch in the cafeteria. It was usually full, so I had to share the table with other NIH investigators, who were always engrossed in a scientific discussion. Thanks to what I learned at the journal club meetings, I was frequently able to understand most of what was being discussed. At that time, I was quite shy, but one day, I dared to ask a question about the conversation. To my astonishment, they not only answered but involved me in their discussion. Even though the conversations lasted no more than twenty min (after which time, the scientists would leave in a hurry, as they all seemed to have bosses who were watching the clocks), I learned enormously from them. Risking a reprimand from the Tabors, I frequently used to have lunch at one table and then, when the other people seated there left in a hurry, I would get my dessert and choose a different table to engage in more conversation. The Tabors never complained about my lunch delays, and on one particular day when I stayed in the cafeteria a bit longer than necessary, Herbert asked me in a humorous tone (meaning no reproach) if I enjoyed my lunch. I babbled something and quickly went back to the bench. The impression remained, however, that the Tabors knew all too well what was going on in the cafeteria and the powerful seduction of the conversation going on there. These are only two examples of the rich atmosphere of the Tabors' laboratory.
      Celia and Herbert were always very solicitous and ready to discuss any kind of results I managed to get, but I was never able to get many. At the end of my fellowship, all I was able to show for my time in their laboratory was a small chapter in Methods in Enzymology, which was written by the Tabors (
      • Tabor H.
      • Tabor C.W.
      • de Meis L.
      Chemical synthesis of N-acetyl-1,4-diaminobutane, N1-acetylspermidine, and N8-acetylspermidine.
      ). This poor productivity was due to my lack of scientific maturity. There were plenty of opportunities in the Tabors' department, but I was not yet ready to benefit from them. However, despite this apparently meager result, it was in the Tabors' laboratory that I really learned what biochemistry was about and became aware of the vast horizon of the biomedical sciences. Back home in Brazil, it was difficult to explain in reports that I had learned a lot at the NIH journal club meetings and cafeteria conversations. As a result, there was a certain disappointment regarding my seeming lack of performance at the NIH. When I left Brazil, I was considered a bright young fellow who had published several papers with Dr. Oswaldo Cruz, and in the United States, land of opportunity, I was expected to blossom and publish much more. It was difficult to explain how important it was for my scientific education to be one of the Tabors' students.
      Shortly after my return from the United States, I obtained a position at the Biophysics Institute of the Federal University of Rio de Janeiro, my alma mater. It was at this time that I married a very attractive and bright geologist. The quality of my life greatly improved after my marriage and migration to the university. In addition to the friendly attitude of the staff, I was allowed to work independently on my own projects. It was at the Biophysics Institute that I became productive, and after two years, I had published four papers in respected international journals. After a long, depressing period without any publications, I had started to doubt my talent; however, with these papers, I proved to myself that I could do it. It was during this period that I stumbled on membrane-bound enzymes and, in particular, on skeletal muscle Ca2+-transport ATPase, a single peptide of 110 kDa that is found in abundance in vesicles derived from the muscle reticulum. This became the main subject of my following working years.
      Two of the four papers I published at that time were related to Ca2+-transport ATPases, also named Ca2+ pumps. The first one described the finding that the brain microsomal fraction contains a Ca2+ pump with the same properties as the muscle Ca2+ pump (
      • de Meis L.
      • Rubin-Altschul M.
      • Machado R.D.
      Comparative data of Ca2+ transport in brain and skeletal muscle microsomes.
      ). At that time, it was thought that the Ca2+ pumps were confined to skeletal muscle, and from then on, different isoforms of Ca2+ pumps were found in different tissues (
      • de Meis L.
      • Rubin-Altschul M.
      • Machado R.D.
      Comparative data of Ca2+ transport in brain and skeletal muscle microsomes.
      ,
      • Wuytack F.
      • Papp B.
      • Verboomen H.
      • Raeymaekers L.
      • Dode L.
      • Bobe R.
      • Enouf J.
      • Bokkala S.
      • Authi K.S.
      • Casteels R.
      A sarco/endoplasmic reticulum Ca2+-ATPase 3-type Ca2+ pump is expressed in platelets, in lymphoid cells, and in mast cells.
      ,
      • Lytton J.
      • Westlin M.
      • Burk S.E.
      • Shull G.E.
      • MacLennan D.H.
      Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.
      ,
      • Cardoso C.M.
      • Rumjanek V.M.
      • de Meis L.
      Uncoupling of Ca2+ transport ATPase in muscle and blood platelets by diacylglycerol analogues and cyclosporin A antagonism.
      ) and renamed sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). In the second work, I showed that acetyl phosphate could be used as a substrate by SERCA. Until then, it had been thought that only ATP could be used as a substrate by SERCA. Later, it was shown that like acetyl phosphate, ITP and GTP could be used as substrates, but their affinity was orders of magnitude smaller than that of ATP. The intriguing finding was that the kinetics of the pump varied depending on whether acetyl phosphate or ATP was used as the substrate (
      • de Meis L.
      Activation Ca2+ uptake by acetyl phosphate in muscle microsomes.
      ,
      • de Meis L.
      Ca2+ uptake and acetyl phosphatase of skeletal muscle microsomes. Inhibition by Na+, K+, Li+, and adenosine triphosphate.
      ,
      • de Meis L.
      • Hasselbach W.
      Acetyl phosphate as substrate for Ca2+ uptake in skeletal muscle microsomes. Inhibition by alkali ions.
      ).

      The Brazilian Military Dictatorship, Acetyl Phosphate, and Heidelberg

      Shortly after my return from the United States in 1964, the military took over the Brazilian government. These were dark years in Brazil. The new political regime selected a new director for the Oswaldo Cruz Institute, a man who did not have the foggiest idea what science was about but claimed to be very loyal to the new regime. One of the loyal director's deeds was to make the working conditions of the few active laboratories simply unbearable. After a year, I resigned and went to the university to teach and do research at the Biophysics Institute in the department headed by Antonio Paes de Carvalho. The institute was directed by Carlos Chagas Filho. In addition to being a good scientist, Chagas Filho had great diplomatic ability. His family was one of the most traditional and influential in the country, and he was able to protect the Biophysics Institute without ever compromising with the military regime.
      At the beginning of the dictatorship, the bad guys were the communists, but they had a peculiar view of communism. Later on, in the 1960s, the bad guys were no longer the communists, but those who did not approve of the military regime were referred to as subversive. I never cared for politics, least of all for communism: I did not approve of the lack of freedom and, above all, if one of my students was arrested and tortured by the regime. My colleagues from the institute and I used to go from prison to prison looking for missing students. If no one searched for them, the regime thought they were of no importance or in fact subversives whom nobody wanted to be involved with and simply made them disappear. The regime did not like the interference of those who went looking for prisoners.
      One day, the president of the Brazilian Academy of Science told me he had heard rumors that my family and I should take a long sabbatical leave. He assured me that he would let me know when things got better and I could come back safely. My wife was a well known geomorphologist. We both wrote letters seeking positions in different places, mostly in the United States. We received several favorable answers, but none in the same place except for Germany, where my wife was accepted at the University of Heidelberg and Professor Hasselbach (Fig. 6) offered me a nice visiting professor position at the Max Planck Institute. This would keep the family together, and I had a chance to work in the laboratory of the discoverer of the sarcoplasmic reticulum Ca2+-transport ATPase (
      • Hasselbach W.
      • Makinose M.
      The calcium pump of the “relaxing granules” of muscle and its dependence on ATP splitting.
      ).
      The work with acetyl phosphate (
      • de Meis L.
      Activation Ca2+ uptake by acetyl phosphate in muscle microsomes.
      ,
      • de Meis L.
      Ca2+ uptake and acetyl phosphatase of skeletal muscle microsomes. Inhibition by Na+, K+, Li+, and adenosine triphosphate.
      ) played an important role in my sojourn in Heidelberg during the military regime's reign in Brazil. In 1970, Hasselbach and his close collaborator, Makinose, were in the process of discovering that the Ca2+-ATPase could use the energy derived from a Ca2+ gradient to synthesize ATP from ADP and Pi (
      • Barlogie B.
      • Hasselbach W.
      • Makinose M.
      Activation of calcium efflux by ADP and inorganic phosphate.
      ,
      • Makinose M.
      Calcium efflux-dependent formation of ATP from ADP and orthophosphate by the membranes of the sarcoplasmic vesicles.
      ,
      • Makinose M.
      • Hasselbach W.
      ATP synthesis by the reverse of the sarcoplasmic calcium pump.
      ). In the course of their experiments, they needed vesicles loaded with Ca2+ in a suspension completely free of ATP and ADP. This was not easy to obtain using ATP to load the vesicles but could be achieved using acetyl phosphate. Years later, I learned from Makinose that my letter asking for a position reached Hasselbach's office shortly after they had read my work on acetyl phosphate, and this apparently contributed to the generous offer I received from Germany.
      When I arrived at the Max Planck Institute, Hasselbach asked me what I would like to work on, and I suggested that I continue exploring the kinetic differences between acetyl phosphate and ATP hydrolysis. Hasselbach said fine, carry on, thus giving me freedom to plan my own experiments. When I had a sufficient amount of data, he would come around two or three days later and discuss the data with me, together with Makinose. I enjoyed my work but noticed that, every two days, Makinose and Hasselbach closed themselves in Makinose's office and had long sotto voce discussions. This was intriguing, but it was none of my business, so I got used to it and did not pay much attention. What I did not know was that the two of them were immersed in experiments that would completely change the prevailing view of energy interconversion.
      They showed that a steep Ca2+ gradient was formed when vesicles loaded with acetyl phosphate were transferred to a medium without Ca2+ and with EGTA, a Ca2+-chelating substance. In these conditions, the Ca2+ retained by the vesicles leaked at a slow rate to the medium. The rate of Ca2+ leaking did not vary if either Pi or a small amount of ADP was added to the medium. However, if both ADP and Pi were added, then Ca2+ was released at a fast rate, and coupled with the increment in Ca2+ release, there was net synthesis of ATP. In 1971, Makinose and Hasselbach published three short papers in FEBS Letters (
      • Barlogie B.
      • Hasselbach W.
      • Makinose M.
      Activation of calcium efflux by ADP and inorganic phosphate.
      ,
      • Makinose M.
      Calcium efflux-dependent formation of ATP from ADP and orthophosphate by the membranes of the sarcoplasmic vesicles.
      ,
      • Makinose M.
      • Hasselbach W.
      ATP synthesis by the reverse of the sarcoplasmic calcium pump.
      ) demonstrating that the Ca2+-ATPase, similar to mitochondrial ATPase, could use the energy derived from an ionic gradient (Ca2+) to synthesize ATP from ADP and Pi. In 1972, Makinose demonstrated that the synthesis of ATP was initiated by phosphorylation of the enzyme by Pi, forming an acyl phosphate residue at the catalytic site (
      • Makinose M.
      Phosphoprotein formation during osmo-chemical energy conversion in the membrane of the sarcoplasmic reticulum.
      ). In accordance with what was described in the Ca2+ uptake, it was concluded that during reversal of the pump, the energy derived from the gradient was captured by the enzyme to form the acyl phosphate residue from Pi (Reactions I and II).
      Figure thumbnail fx1
      Figure thumbnail fx2
      These observations had an important impact on the bioenergetics field because they showed that in addition to mitochondria, other proteins could convert osmotic into chemical energy and that the interconversion could be catalyzed by a simple protein, a monomer with a molecular mass of 110 kDa. Because the mitochondrial F1F0 complex was a large polymer with many subunits of different molecular mass, it was thought that interconversion of osmotic into chemical energy required complex protein structures. At present, it is clear that the ability to interconvert these forms of energy is not related to the complexity of the enzyme. However, at the time of Mitchell, Hasselbach, and Makinose, these were the subjects of clamorous discussions in bioenergetics meetings.
      I was fascinated by these new findings when Makinose told me about them a few days before publication. I could hardly believe that such important experiments were being discussed when Hasselbach went to Makinose's office.
      We had a great time in Heidelberg, a small, lovely city crossed by the peaceful Neckar River. It contained quaint features: a castle on a hill; a romantic old bridge; and a happy section called the “Old City” that contained all sorts of small restaurants, stores selling exotic items, and lots of tourists. The institute directed by Hasselbach had a small administrative staff and practically no bureaucracy. It used to close on the weekends and was practically empty during week days after 6 p.m. However, the rest of the time, the work was intensive, disciplined, and productive.
      The high point of my stay was the intimate relationships I formed with both Makinose and Hasselbach, in part due to music. Makinose played the violin and had two nieces who studied music at the university. I used to play the recorder reasonably well. Once a week, we met at Makinose's house after dinner and played for one hour, after which Makinose's wife would bring us snacks and a delicious tea. On the other hand, Hasselbach disliked music, at least our kind of music, but his wife enjoyed playing the recorder. She invited me to play and have a glass of wine once every two weeks at their home, which I was happy to do. The music sessions lasted less than half an hour, at which time Hasselbach would come in and state firmly that we had had enough music. He would then take me to the garden, where he would serve a very tasty Rhine wine, and we would become engrossed in marvelous conversations, in which I learned a lot about both science and life. I praised Frau Hasselbach's music skills, but I confess that what I really enjoyed was the wine and the highly inspiring conversation with Hasselbach. When my contract with the Max Planck Institute expired, Hasselbach offered me a permanent position at the institute with a higher salary. It was tempting, but I had just received letters from the president of the Brazilian Academy of Science and various colleagues at the university informing me that the situation in Brazil had improved greatly and that there was money available for research. Thus, I declined Hasselbach's excellent offer and returned home, where I was warmly received back at the Biophysics Institute. Before leaving Heidelberg, I asked Hasselbach and Makinose if they would mind if I followed the exciting new path they had discovered. Both of them warmly agreed, and for several years, most of my work focused on the reversal of the Ca2+ pump. As far as I know, unlike the ability to remove Ca2+ from the cytosol, there is no clear-cut evidence that the reversal of the pump has an important role in cell physiology. However, the pump reversal proved to be an excellent tool to study energy transduction, and through the reversal, it was possible to reveal the various steps of the enzyme catalytic cycle working both forward and backward. In addition, it provided information about the concept of high- and low-energy phosphate compounds.

      How a Small Technical Detail Can Lead to Different Interpretations

      In 1973, it was known that sarcoplasmic reticulum vesicles contain a small amount of endogenous Ca2+. It was then hypothesized that if empty vesicles were suspended in a medium with EGTA, a small gradient should be formed across the sarcoplasmic reticulum vesicles due to the small Ca2+ inside the vesicles, and this should lead to a small enzyme phosphorylation by Pi. This possibility was simultaneously investigated in the laboratory of Paul Boyer and in my own laboratory. In 1973, Kanazawa and Boyer found that when empty vesicles were solubilized with the detergent Triton X-100 and incubated in a medium containing EGTA, Pi, and Mg2+, a temporary, small amount of phosphoenzyme was formed (
      • Kanazawa T.
      • Boyer P.D.
      Occurrence and characteristics of a rapid exchange of phosphate oxygens catalyzed by sarcoplasmic reticulum vesicles.
      ). This was orders of magnitude smaller than that measured by Makinose with Ca2+-loaded vesicles. The low Pi phosphorylation was abolished rapidly (in seconds) when the SERCA vesicles were disrupted with the detergent Triton X-100. These data led the authors to conclude that the small gradient formed with the endogenous Ca2+ could indeed lead to a small phosphorylation, which was abolished with the dissipation of the microgradient after a short incubation time, meaning that the presence of a gradient was an absolute requirement for the phosphorylation of the enzyme by Pi.
      In Rio de Janeiro, we performed experiments similar to those of Kanazawa and Boyer (FIGURE 7, FIGURE 8), but instead of using Triton X-100, we permeabilized the vesicles with a small amount of ethyl ether, which was known to disrupt the vesicles' membrane (
      • Masuda H.
      • de Meis L.
      Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions.
      ,
      • de Meis L.
      • Masuda H.
      Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate through two different reactions.
      ). With the leaky vesicles and in the absence of Ca2+ (excess EGTA), we measured phosphorylation of the Ca2+-ATPase to a level that was smaller than that measured by Makinose but was an order of magnitude higher than that measured by Kanazawa and Boyer. An important feature was that the phosphoenzyme was stable and did not decrease even after a period of 30 min in the presence of excess EGTA. This indicated that, in contrast to the results of Kanazawa and Boyer, the Ca2+-ATPase could be phosphorylated by Pi in the absence of a gradient, i.e. in the absence of an apparent source of energy. We then decided to vary the experimental conditions in an attempt to increase the level of stable phosphoenzyme measured in the absence of a gradient, and the best results were attained when the pH of the medium was decreased from 7.0 to a range of 5.5–6.0 and the Pi concentration in the medium was raised from 1–2 mm to a range of 5–8 mm. Under these conditions, we could obtain the same level of phosphoenzyme as that measured with the gradient by Makinose (
      • Makinose M.
      Phosphoprotein formation during osmo-chemical energy conversion in the membrane of the sarcoplasmic reticulum.
      ). Different tests confirmed that the acyl phosphate formed in the absence of the gradient was the very same aspartyl phosphate residue as that formed with the intact vesicles and gradient. It was known that the Ca2+-ATPase could be solubilized with Triton X-100 and remained stable for hours provided that Ca2+ (0.1 mm) was included in the medium. Later, Kanazawa (Fig. 8) showed that the Ca2+-ATPase was quickly denatured if solubilized with Triton X-100 in the absence of Ca2+ (
      • Kanazawa T.
      Phosphorylation of solubilized sarcoplasmic reticulum by orthophosphate and its thermodynamic characteristics. The dominant role of entropy in the phosphorylation.
      ). This explained the earlier findings obtained with Boyer, which gave the incorrect impression that there was no phosphorylation by Pi in the absence of a gradient. We were lucky to permeate the vesicles with ethyl ether, a treatment that does not damage the Ca2+-ATPase. Otherwise, we also would have missed the phosphorylation by Pi in the absence of a Ca2+ gradient.
      Figure thumbnail gr8
      FIGURE 8Kanazawa in Rio de Janeiro. He showed us the deleterious effects of the detergent Triton X-100 on sarcoplasmic vesicles when Ca2+ was removed from the medium with an excess of the Ca2+-chelating agent EGTA.
      Eventually, Paul Boyer came to Rio, and we repeated the experiment with leaky vesicles (Fig. 7). Paul was convinced that in fact phosphorylation could occur without a gradient. I learned a lot from Paul's sojourn and started to admire him not only because of his excellence in science but also for his kind and unassuming personality. During his visit, Paul explained to us his recent Pi ↔ HO18H exchange experiments with mitochondria indicating that ATP could be synthesized spontaneously at the catalytic site of the mitochondrial F1-ATPase without the need for energy. The amount of ATP synthesized was very small and tightly bound and could not dissociate from the enzyme. Thus, its formation could only be inferred from Pi ↔ HO18H exchange measurements. According to Paul's findings, first published in 1973, energy was needed not for the synthesis of ATP but for the dissociation of the tightly bound ATP from the enzyme (
      • Boyer P.D.
      • Cross R.L.
      • Momsen W.
      A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions.
      ). After Paul's return to the United States, we started to collaborate from a distance, measuring Pi ↔ HO18H exchange catalyzed by the Ca2+-ATPase under different conditions. The experiments were performed in Rio and the analysis of O18 in Los Angeles. In 1997, Paul was awarded the Nobel Prize for the discovery of the mechanism through which the mitochondrial ATP synthase works.
      Figure thumbnail gr7
      FIGURE 7Paul Boyer during his visit to Rio de Janeiro. Paul and I together repeated the experiments with leaky vesicles.

      The ATP ↔ Pi Exchange Reaction and the Low-affinity Ca2+-binding Site

      Phosphorylation of the Ca2+-ATPase by Pi is very sensitive to the presence of Ca2+ in the medium regardless of whether the vesicles are leaky or loaded with Ca2+. The Ca2+ concentration needed in the assay medium for half-maximal inhibition is 1 μm (
      • Masuda H.
      • de Meis L.
      Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions.
      ). Unlike Pi, phosphorylation of the Ca2+-ATPase by ATP requires the presence of Ca2+ in the medium (

      Makinose, M., (1966) Abstracts of the Second International Congress of Biophysics, Vienna, No. 276,.

      ,
      • Makinose M.
      The phosphorylation of the membranal protein of the sarcoplasmic vesicles during active calcium transport.
      ). Coincidentally, the Ca2+ concentration needed for half-maximal phosphorylation by ATP was found to be the same as that needed to inhibit phosphorylation by Pi, indicating that the binding of Ca2+ to a single binding site located in the outer surface of the vesicles determines whether the enzyme will be phosphorylated by ATP or by Pi (
      • de Meis L.
      • Masuda H.
      Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate through two different reactions.
      ,
      • de Meis L.
      • Vianna A.L.
      Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum.
      ,
      • de Meis L.
      ). Despite the fact that the enzyme can be phosphorylated by Pi in the presence and absence of a gradient, there is a major difference between the two forms: the ability to synthesize ATP. Although the vesicles loaded with Ca2+ were able to transfer their phosphate to ADP, forming ATP, the phosphoenzyme formed in the absence of a gradient was not able to synthesize ATP. The details of the transference of phosphate to ADP will be discussed in further detail below, but this observation led to the following two conclusions.
      (i) The same chemical species, an acyl phosphate, can be of high or low energy. When the concept of phosphate compounds of high and low energy was formulated by Lipmann in 1941 (
      • Lipmann F.
      Metabolic generation and utilization of phosphate bond energy.
      ), it was thought that the energy level of a phosphate residue would always be the same, and on the basis of the data available at that time, the possibility that the energy of hydrolysis might change in the surface of an enzyme was not taken into consideration. (ii) At least for the Ca2+-ATPase, the energy derived from the gradient (osmotic) is not used for the formation of the acyl phosphate bond at the catalytic site but is needed to convert it from low to high energy.
      The ATP ↔ Pi exchange reaction was first described in mitochondria when a proton gradient is formed across the membrane. During the exchange, mitochondria are able to hydrolyze and synthesize ATP simultaneously, i.e. although some units of the ATP synthase cleave ATP, other units synthesize ATP from ADP and Pi using the energy derived from the H+ gradient. Inspired by the mitochondrial ATP ↔ Pi exchange, in 1971, Makinose observed that the Ca2+-ATPase also can catalyze the simultaneous hydrolysis and synthesis of ATP when the vesicles are filled with Ca2+ and a steady state between Ca2+ influx and Ca2+ efflux is reached (
      • Makinose M.
      Calcium efflux-dependent formation of ATP from ADP and orthophosphate by the membranes of the sarcoplasmic vesicles.
      ). Because of the need for an ionic gradient, the ATP ↔ Pi exchange reaction was one of the parameters used to characterize the interconversion between osmotic and chemical energy. In fact, the disruption of the muscle vesicle integrity with either phospholipase A or diethyl ether abolished the synthesis of ATP, and the Ca2+-ATPase was only able to cleave the ATP available in the medium. This led to the conclusion that at steady state, the energy derived from the hydrolysis of ATP was used to maintain the Ca2+ gradient, and at the same time, the energy derived from the gradient was used to synthesize ATP from ADP and Pi.
      The general view of Ca2+ transport was that the ion bound in a part of the transport ATPase facing the outer surface of the vesicles, and the energy derived from the cleavage of ATP was then used to promote a conformational change in the protein that would permit the release of the ion into the vesicle lumen. Thus, in physiological conditions, when a gradient is formed, the Ca2+ concentration in the solution facing the outer surface of the vesicle is very low, whereas inside the vesicle, it reaches a range of 2–10 mm. We then raised the possibility that the conversion of the phosphoenzyme from low to high energy could be related to the binding of Ca2+ to a low-affinity site of the ATPase facing the vesicle lumen. Thus, the conversion from low to high energy was related to Ca2+ binding (binding energy) and not to the gradient itself (osmotic energy). To test this hypothesis, we prepared leaky vesicles and incubated them in medium containing various Ca2+ concentrations from the range similar to that found on the outer surface of the vesicles during transport (submicromolar) up to the range found in the vesicle lumen during Ca2+ accumulation (millimolar). The result of this experiment was very rewarding. In the presence of low Ca2+ concentrations, the ATPase was only able to catalyze the hydrolysis of ATP, but as the Ca2+ concentration was raised to the millimolar range, there was a simultaneous inhibition of the hydrolysis of ATP and the activation of ATP synthesis. The Ca2+ concentration needed for both half-maximal inhibition of ATP cleavage and half-maximal activation of ATP synthesis was in the range of 1–2 mm (
      • de Meis L.
      • Vianna A.L.
      Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum.
      ,
      • de Meis L.
      ,
      • de Meis L.
      • Costa Carvalho Mda G.
      Role of the Ca2+ concentration gradient in the adenosine 5′-triphosphate-inorganic phosphate exchange catalyzed by sarcoplasmic reticulum.
      ). These experiments supported the hypothesis that the synthesis of ATP was promoted by the asymmetrical binding of Ca2+ on the two sides of the membrane and not by the osmotic energy derived from the gradient.

      The Reaction Sequence

      The data accumulated from the studies of the pump working forward (ATP hydrolysis) and backward (ATP synthesis) led us to the proposal of a basic catalytic cycle shown in Fig. 9A. During catalysis, the enzyme cycles through two distinct conformations, E1 and E2 (originally E and *E) (
      • de Meis L.
      • Vianna A.L.
      Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum.
      ,
      • de Meis L.
      ,
      • Carvalho M.G.
      • de Souza D.G.
      • de Meis L.
      On a possible mechanism of energy conservation in sarcoplasmic reticulum membrane.
      ,
      • de Meis L.
      Role of water in the energy of hydrolysis of phosphate compounds: energy transduction in biological membranes.
      ,
      • Tanford C.
      Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump.
      ). The enzyme form E1 binds calcium with high affinity (Ks ∼ 1 μm) on the outer surface of the vesicles and can be phosphorylated by ATP (Fig. 9, reactions 1 and 2) but not by Pi. This reaction has a Keq value of ∼1 and is therefore fully reversible (
      • de Meis L.
      • Tume R.K.
      A new mechanism by which an H+ concentration gradient drives the synthesis of adenosine triphosphate, pH jump, and adenosine triphosphate synthesis by the Ca2+-dependnet adenosine triphosphatase of sarcoplasmic reticulum.
      ). The ATPase undergoes a conformational change from E1∼P to E2-P (reaction 3). During this transition, Ca2+ remains bound to the enzyme but now faces the luminal side of the vesicle. The Ca2+ affinity of the enzyme form E2 is ∼3 orders of magnitude smaller than that of E1 (∼1–2 mm). During Ca2+ dissociation (reaction 4), the energy of hydrolysis of the phosphoenzyme decreases to practically zero (Keq = 1) and becomes fully reversible, thus explaining why the ATPase can be phosphorylated by Pi in the absence of a gradient (reaction 5, reverse direction). Two Ca2+ ions are bound to each enzyme unit, and the energy required for the decrease in the Ca2+ Ks from 10−6 to 10−3 m is about −18 kcal/mol. This is in the range of the energy derived from ATP hydrolysis. This value was confirmed later in calorimetric experiments (see “Thermogenesis: Heat Production by the Ca2+-ATPase ” below). The finding of phosphoenzyme of low and high energy indicates that the energy derived from ATP is not released at the time of phosphoenzyme cleavage, but it is captured by the enzyme before the cleavage is completed. In this case, binding energy and conformational energy are involved. It is as if the enzyme sucks the energy from the phosphoenzyme and converts it into work before being cleaved (reactions 3 and 4).
      Figure thumbnail gr9
      FIGURE 9Catalytic cycle of the Ca2+-ATPase. A, the sequence includes two distinct enzyme conformations, E1 and E2. The Ca2+-binding sites in the E1 form face the external surface of the vesicle and have a high affinity for Ca2+ (Ka = 10−6 m at pH 7). In the E2 form, the Ca2+-binding sites face the vesicle lumen and have a low affinity for Ca2+ (Ka = 10−3 m at pH 7). The enzyme form E1 is phosphorylated by ATP but not by Pi, forming the high-energy phosphoenzyme 2Ca:E1∼P (Keq hydrolysis of ∼106 and ΔG0 ∼ −8.4 kcal/mol). The enzyme form E2 is phosphorylated by Pi but not by ATP, forming the low-energy phosphoenzyme Ca:E2-P (reaction 5; Keq hydrolysis of ∼1 and ΔG0 ∼ 0 kcal/mol). When the Ca2+ concentration on the two sites of the membrane is <50 μm (leaky vesicles), reaction 4 is irreversible, and this forces the sequence to flow forward from reactions 1 to 6. When the Ca2+ concentration in the vesicle lumen rises above 1 mm, the catalytic cycle is reversed from reactions 5 to 2, and the Ca2+ efflux is coupled with ATP synthesis. These steps were described by our laboratory (
      • de Meis L.
      • Vianna A.L.
      Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum.
      ). B, reaction 7 was described by the Inesi laboratory (
      • Yu X.
      • Inesi G.
      Variable stoichiometric efficiency of Ca2+ and Sr2+ transport by the sarcoplasmic reticulum ATPase.
      ,
      • Fortea M.I.
      • Soler F.
      • Fernandez-Belda F.
      Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP.
      ) as was referred to as Ca2+ slippage. Later, we showed that this step regulates the amount of work and heat released during ATP hydrolysis (
      • de Meis L.
      Ca2+-ATPases (SERCA): energy transduction and heat production in transport ATPases.
      ).
      This cycle has been confirmed in different laboratories (
      • de Meis L.
      Role of water in the energy of hydrolysis of phosphate compounds: energy transduction in biological membranes.
      ,
      • Tanford C.
      Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump.
      ,
      • Toyoshima C.
      • Mizutani T.
      Crystal structure of the calcium pump with a bound ATP analogue.
      ), and the equilibrium constants (Keq) of the intermediate steps have been determined by transient kinetics in our laboratory as well as several others. Recently, the structures of the two monomeric states (E1 and E2) have been resolved by x-ray refraction by Toyoshima and Mizutani (
      • Toyoshima C.
      • Mizutani T.
      Crystal structure of the calcium pump with a bound ATP analogue.
      ). The structures obtained indicate that the region of the catalytic site forms a concavity that undergoes a dramatic change in the transition of E1 into E2.

      Net Synthesis of ATP

      Under the conditions used to measure ATP ↔ Pi exchange, there is no net synthesis of ATP; the rate of ATP hydrolysis is always faster than the rate of ATP synthesis. A year after our reports on the ATP ↔ Pi exchange in the absence of a gradient, Knowles and Racker (
      • Knowles A.F.
      • Racker E.
      Formation of adenosine triphosphate from Pi and adenosine diphosphate by purified Ca2+-adenosine triphosphatase.
      ) confirmed the formation of the low-energy phosphoenzyme in leaky vesicles at pH 5.5–6.0, and using the concept of asymmetrical Ca2+ binding (
      • de Meis L.
      • Costa Carvalho Mda G.
      Role of the Ca2+ concentration gradient in the adenosine 5′-triphosphate-inorganic phosphate exchange catalyzed by sarcoplasmic reticulum.
      ), they were able to promote the net synthesis of ATP after a single catalytic cycle of the enzyme flowing back in the direction of the reversal of the Ca2+ pump. This was achieved using leaky vesicles and a two-step procedure in which, initially, the enzyme was phosphorylated by Pi in the absence of Ca2+, and then ADP and 3–4 mm CaCl2 were added rapidly to the medium, a procedure referred to as a “jump.” After the Ca2+ jump, half of the phosphoenzyme phosphate was transferred to ADP, forming ATP. Shortly after, we confirmed this experiment using the transient kinetics technique (
      • de Meis L.
      • Tume R.K.
      A new mechanism by which an H+ concentration gradient drives the synthesis of adenosine triphosphate, pH jump, and adenosine triphosphate synthesis by the Ca2+-dependnet adenosine triphosphatase of sarcoplasmic reticulum.
      ).

      Solvation Energy and Phosphate Compounds of High and Low Energy

      In 1970, George et al. (
      • George P.
      • Witonsky R.J.
      • Trachtman M.
      • Wu C.
      • Dorwart W.
      • Richman L.
      • Richman W.
      • Shurayh F.
      • Lentz B.
      “∼H2O.” An enquiry into the importance of solvation effects in phosphate ester and anhydride reactions.
      ) proposed a new theory for the concept of high-energy (∼Pi) and low-energy (–P) phosphates. The working title of the article was “∼H2O,” and in it, the authors proposed that the energy of hydrolysis of the phosphate compound (Keq) was determined by the solvation energies of reactant and products. Solvation energy is the amount of energy needed to remove the solvent molecules that organize around a molecule in solution. Later, Hayes et al. (
      • Hayes D.M.
      • Kenyon G.L.
      • Kolman P.A.
      Theoretical calculations of the hydrolysis energies of some “high-energy” molecules. 2. A survey of some biologically important hydrolytic reaction.
      ) calculated the energy of hydrolysis of several phosphate compounds in the gas phase, i.e. in an aqueous medium but in a theoretical condition in which the solvent would not bind to the reactants. The values found were quite different from those measured in water, thus supporting the proposal of George et al. (
      • George P.
      • Witonsky R.J.
      • Trachtman M.
      • Wu C.
      • Dorwart W.
      • Richman L.
      • Richman W.
      • Shurayh F.
      • Lentz B.
      “∼H2O.” An enquiry into the importance of solvation effects in phosphate ester and anhydride reactions.
      ). According to these calculations, the energy of hydrolysis of an acyl phosphate residue solvated in water would be in the range of −8 to −10 kcal/mol but increased to +5 to +32.5 kcal/mol in the gas phase. We then reasoned that the interaction of water with the reactant should decrease if we decreased the water activity of the solvent, thus mimicking the gas phase. We then raised the possibility that in the E2 conformation, the catalytic site of the enzyme would be hydrophobic and that the solvation of both Pi and the aspartate residue would decrease to a point that phosphorylation would occur spontaneously (E2-P). In this case, the major thermodynamic barrier for the formation of the acyl phosphate residue would be not the formation of the covalent bond (Fig. 9A, reaction 5) but the binding of Pi to the enzyme, i.e. the partitioning of a hydrophilic ion (Pi) from the aqueous assay medium into the hydrophobic environment of the catalytic site. Factors facilitating this partition should also facilitate the phosphorylation of the enzyme by Pi. The phosphoenzyme E2-P formed from Pi would not be able to transfer its phosphate to ADP because of the large difference in the energies of hydrolysis of the acyl phosphate in a hydrophobic environment and ATP in an aqueous solution. The binding of Ca2+ to the low-affinity site of the enzyme would then promote a conformational change in the protein that would allow the entry of water into the catalytic site with the subsequent solvation of both the acyl phosphate residue and ADP. As a result, the energy values for the hydrolysis of the acyl phosphate and ATP would become equally high, and ATP synthesis would proceed spontaneously. Experimental conditions that reduce the entry of water into the catalytic site should also impede the synthesis of ATP. According to this hypothesis, the existence of high- and low-energy forms of the phosphoenzyme would be related solely to the water activity in the catalytic site. We tested this hypothesis by measuring the phosphorylation of the enzyme by Pi and the synthesis of ATP in the presence of water and various concentrations of nondenaturing organic solvents such as dimethyl sulfoxide, glycerol, and N,N-dimethylformamide (
      • de Meis L.
      Role of water in the energy of hydrolysis of phosphate compounds: energy transduction in biological membranes.
      ,
      • de Meis L.
      • Martins O.B.
      • Alves E.W.
      Role of water, hydrogen ion, and temperature on the synthesis of adenosine triphosphate by the sarcoplasmic reticulum adenosine triphosphatase in the absence of a calcium ion gradient.
      ,
      • de Meis L.
      Ca2+-ATPases (SERCA): energy transduction and heat production in transport ATPases.
      ,
      • de Meis L.
      • Inesi G.
      ATP synthesis by sarcoplasmic reticulum ATPase following Ca2+, pH, temperature, and water activity jumps.
      ). In aqueous mixtures, these solvents markedly increase the partition coefficient of Pi from the aqueous medium into an organic phase containing isobutyl alcohol and benzene. In previous studies, we observed that in the absence of a gradient, the phosphorylation of E2 by Pi demonstrated a saturation behavior (
      • Masuda H.
      • de Meis L.
      Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions.
      ,
      • de Meis L.
      • Masuda H.
      Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate through two different reactions.
      ,
      • de Meis L.
      ,
      • Tanford C.
      Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump.
      ), indicating the occurrence of a phosphate-enzyme complex prior to the phosphorylation reaction (Reaction III).
      Figure thumbnail fx3
      If the catalytic site of the enzyme is hydrophobic, then the partitioning of Pi from the assay medium into the catalytic site (k1/k−1) should be facilitated when the difference in hydrophobicity between the two compartments is decreased by the addition of organic solvent to the medium, and this should promote a decrease in the apparent Km for Pi. Accordingly, after replacing 40% of the water in the assay medium with dimethyl sulfoxide, we observed that the pronounced pH dependence was abolished and that the apparent Km of E2 for Pi decreased by up to a thousandfold (
      • de Meis L.
      Ca2+-ATPases (SERCA): energy transduction and heat production in transport ATPases.
      ). However, the phosphoenzyme formed in the presence of 40% dimethyl sulfoxide was not converted into a high-energy form after the addition of ADP and a Ca2+ concentration sufficient to bind to the low-affinity site of the enzyme, i.e. there was no synthesis of ATP after the addition of Ca2+ and ADP to the medium. The inhibition of ATP synthesis was related to the decrease in water activity caused by the organic solvent because the synthesis of ATP was restored if, after the addition of ADP and Ca2+, the dimethyl sulfoxide concentration was suddenly decreased from 40 to 2%. The hypothesis proposed states that high- and low-energy forms of the phosphoenzyme are correlated with the availability of water at the catalytic site of the enzyme. The experimental results were consistent with this hypothesis. As predicted, phosphorylation of the enzyme by Pi was facilitated when the hydrophobicity of the medium was increased by the organic solvents, and after the addition of Ca2+, the phosphoenzyme was only able to transfer its phosphate to ADP if the water activity of the medium was increased by dilution of the organic solvent with water. These data were reproduced in different laboratories and extended to other enzymes such as the mitochondrial F1-ATPase (
      • de Meis L.
      Role of water in the energy of hydrolysis of phosphate compounds: energy transduction in biological membranes.
      ,
      • Sakamoto J.
      Effect of dimethylsulfoxide on ATP synthesis by mitochondrial soluble F1-ATPase.
      ,
      • Kandpal R.P.
      • Stempel K.E.
      • Boyer P.D.
      Characteristics of the formation of enzyme-bound ATP from medium inorganic phosphate by mitochondrial F1-adenosine triphosphatase in the presence of dimethyl sulfoxide.
      ) and inorganic pyrophosphatase (
      • de Meis L.
      • Behrens M.I.
      • Celis H.
      • Romero I.
      • Gómez Puyou M.T.
      • Gómez Puyou A.
      Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient.
      ). In these two systems, the addition of 40% dimethyl sulfoxide promoted both a large decrease in the apparent Km for Pi and a substantial increase in the equilibrium level of the tightly bound ATP (soluble mitochondrial F1-ATPase) and the level of the PPi synthesized by the pyrophosphatase. Finally, the recently reported crystal structure of the Ca2+-ATPase indicates that the conversion of the E1 form into the E2 form involves structural changes in the region of the catalytic site that are compatible with the hydrophobic-hydrophilic transition proposed for the conversion of the phosphoenzyme from low to high energy (
      • Toyoshima C.
      • Mizutani T.
      Crystal structure of the calcium pump with a bound ATP analogue.
      ).

      Can SERCA1 Interconvert Other Forms of Energy Such as pH Gradients and Temperature?

      The Ca2+ affinity of the two enzyme forms E1 and E2 varies with the pH of the medium. Both Ca2+-binding sites exhibited an increased affinity for Ca2+ at high pH and a reduced affinity at lower pH values, with the relative differences in affinities of the two sites remaining unchanged at all pH values. Based on this finding, the following rationale was developed. No ATP synthesis will be observed when the same Ca2+ concentration exists on each side of the membrane and at a level sufficient to saturate the high-affinity site but not sufficient to allow significant binding of Ca2+ to the low-affinity site (Fig. 9A, reversal of reaction 4). However, this system is potentially capable of synthesizing ATP if the Ca2+ affinity of the two binding sites is modified by varying the pH on the two sides of the membrane: alkaline inside the vesicles and acidic in the assay medium. In such a situation, the phosphorylation of the enzyme by Pi will no longer be inhibited, and the Ca2+ concentration could become sufficient to allow the transfer of phosphate to ADP. At pH 5, the level of E2-P is smaller than that measured at pH 6.0. However, at pH 5.0, the Ca2+ concentration needed in the medium for 50% inhibition of E2-P formation is in the range of 1–2 mm Ca2+. The transfer of phosphate from E2-P to ADP is dependent upon the saturation of a low-affinity Ca2+-binding site. After phosphorylation by Pi, it is possible to transfer the phosphate to ADP at progressively lower Ca2+ concentrations as the pH is raised from 6.0 to 8.1. Maximal ATP synthesis at pH 8.1 is attained at a Ca2+ concentration range of 0.5–1 mm. When E2 is phosphorylated at pH 5.0 in the presence of 0.6 mm Ca2+, net synthesis of ATP is observed if, at the time of ADP addition, the pH is rapidly increased to 8.0. These measurements indicate that it is possible to promote the synthesis of ATP after a single catalytic cycle without changing the Ca2+ concentration but by modifying the Ca2+ affinity of the enzyme forms E1 and E2. The measurements also sustain the hypothesis that the sarcoplasmic reticulum vesicles can drive the synthesis of ATP when a 3-pH unit gradient is formed across the vesicle membrane provided that Ca2+ is present in the medium in equal concentrations on the two sides of the membrane (no Ca2+ gradient). In absence of Ca2+, the pH transition does not promote ATP synthesis. A similar procedure can be used by varying rapidly the temperature of the assay medium. The equilibrium of the enzyme forms E1 and E2 and their respective Ca2+ affinities vary significantly with the assay medium temperature. These findings raised the possibility that the Ca2+-ATPase can use the energy derived from either a pH or thermal gradient (
      • Carvalho M.G.
      • de Souza D.G.
      • de Meis L.
      On a possible mechanism of energy conservation in sarcoplasmic reticulum membrane.
      ,
      • de Meis L.
      • Martins O.B.
      • Alves E.W.
      Role of water, hydrogen ion, and temperature on the synthesis of adenosine triphosphate by the sarcoplasmic reticulum adenosine triphosphatase in the absence of a calcium ion gradient.
      ,
      • de Meis L.
      • Inesi G.
      ATP synthesis by sarcoplasmic reticulum ATPase following Ca2+, pH, temperature, and water activity jumps.
      ,
      • Meltzer S.
      • Berman M.C.
      Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum.
      ).

      Pyrophosphate of High and Low Energy

      A direct way to study the role of the solvent in the energy of hydrolysis of a phosphate compound was to measure the Keq and ΔG0 in vitro using media with different water activities. These measurements can substantiate both the theoretical calculation for the gas phase and the assumption for the energy change at the catalytic site of the Ca2+-ATPase. The water molecules that organize around a protein in solution have properties that are different from those of the bulk water medium, e.g. a lower vapor pressure, a lower mobility, and a greatly reduced freezing point. Similar changes in the properties of water are observed in mixtures of solvents and water. The simplest known high-energy phosphate compound is PPi. Thus, we temporarily changed our focus from the Ca2+-ATPase to PPi, aiming to understand better the role of solvation energy. In a totally aqueous medium, the ΔG0 of PPi hydrolysis varies between -3.5 and −4.0 kcal/mol. We found that the energy of hydrolysis of PPi decreased when different organic solvents were included in the medium, reaching ΔG0 values of +2.0 kcal/mol, i.e. it is possible to convert PPi from high energy (ΔG0 = −4.0) to low energy (ΔG0 = +2.0), and this can be achieved by simply modifying the water activity of the medium (
      • de Meis L.
      Pyrophosphate of high and low energy. Contributions of pH, Ca2+, Mg2+, and water to free energy of hydrolysis.
      ,
      • de Meis L.
      • Behrens M.I.
      • Petretski J.H.
      • Politi M.J.
      Contribution of water to free energy of hydrolysis of pyrophosphate.
      ,
      • Behrens M.I.
      • de Meis L.
      Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures.
      ,
      • Romero P.J.
      • de Meis L.
      Role of water in the energy of hydrolysis of phosphoanhydride and phosphoester bonds.
      ). In Boyer's laboratory, it was also shown that the energy of hydrolysis of PPi varies greatly depending on whether it is in solution or bound to the yeast inorganic pyrophosphatase. In the presence of organic solvent, it was possible to decrease the energy of hydrolysis of PPi to values even lower than those measured on the surface of inorganic pyrophosphatase (
      • Janson C.A.
      • Degani C.
      • Boyer P.D.
      The formation of enzyme-bound and medium pyrophosphate and the molecular basis of the oxygen exchange reaction of yeast inorganic pyrophosphatase.
      ). After these findings, I felt much more confident about our proposal that the change in energy of hydrolysis that occurs during the process of energy transduction was indeed related to a change in solvent structure around reactants and products at the catalytic site of enzymes. Thus, I decided to study other enzymes besides the Ca2+-ATPase. The aim was to see if the correlation between water activity and changing energies of hydrolysis at the catalytic site could be extended to different enzymes involved in energy transduction.

      Role of Water Activity in the Process of Energy Transduction by Different Enzymes

      The chromatophores of the photosynthetic bacteria Rhodospirillum rubrum retain a membrane-bound inorganic pyrophosphatase that is able to catalyze both the synthesis and hydrolysis of PPi. When illuminated, the chromatophores use the energy derived from light to form a proton gradient across the membrane, and the membrane-bound pyrophosphatase uses the energy derived from the gradient to catalyze the synthesis of pyrophosphate from inorganic phosphate. With Armando and Marietta Gómez Puyou (Fig. 10), we were able to show that (i) like the Ca2+-ATPase, the R. rubrum inorganic pyrophosphatase is able simultaneously to catalyze the synthesis and hydrolysis of PPi in the absence of a proton gradient; (ii) organic solvent promotes a decrease in the apparent Km of the pyrophosphatase for Pi; and (iii) with purified soluble pyrophosphatase, it was possible to mimic the PPi synthesis of the chromatophores in the light solely by changing the water activity of the medium (
      • de Meis L.
      • Behrens M.I.
      • Celis H.
      • Romero I.
      • Gómez Puyou M.T.
      • Gómez Puyou A.
      Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient.
      ).
      Figure thumbnail gr10
      FIGURE 10Armando (left) and Marietta (right) Gómez Puyou, distinguished scientists from the Universidad Nacional Autónoma de México (UNAM).
      After our studies on the effect of organic solvents on the Ca2+-ATPase, Sakamoto (
      • Sakamoto J.
      Effect of dimethylsulfoxide on ATP synthesis by mitochondrial soluble F1-ATPase.
      ) reported that the affinity of the soluble mitochondrial F1-ATPase for Pi increased by several orders of magnitude when part of the water of the medium was replaced with dimethyl sulfoxide. As a result, there was a increase of several orders of magnitude in the formation of the tightly bound ATP, and this could now be easily measured using 32P. In the absence of organic solvent, the presence of the ATP attached to the enzyme could only be inferred from HOH ↔ O18 measurements. Sakamoto's findings were reproduced in different laboratories, including that of Gómez Puyou (
      • Gómez Puyou A.
      • Tuena de Gómez Puyou M.
      • de Meis L.
      Synthesis of ATP by soluble mitochondrial F1-ATPase and F1-inhibitor-protein complex in the presence of organic solvents.
      ), with whom I had the privilege of collaborating. These findings indicated that, as proposed by us for the Ca2+-ATPase, during oxidative phosphorylation, the catalytic site of mitochondrial ATP synthase undergoes a hydrophobic-hydrophilic transition. In his 1997 Nobel Prize lecture, Boyer quoted our proposal as “the probable mechanism that promotes the spontaneous synthesis of the tightly bound ATP” (
      • Boyer P.D.
      Energy, life, and ATP.
      ).
      Embedded in the plasma membrane of yeast is an H+-ATPase that uses the energy derived from ATP hydrolysis to pump H+ across the membrane. The catalytic cycle of this enzyme is similar to that of the Ca2+-ATPase. Andre Goffeau, an expert on the yeast H+ pump, is one of my best friends (Fig. 11), and by my good luck, he visited my laboratory several times. Together, we found that like the Ca2+-ATPase, the yeast H+-ATPase is able to catalyze the synthesis of ATP (ATP ↔ Pi exchange) in the absence of an H+ gradient and that a decrease in the water activity of the medium leads to both a decrease in the apparent Km for Pi and a severalfold increase in the rate of ATP synthesis (
      • de Meis L.
      • Blanpain J.P.
      • Goffeau A.
      Pi in equilibrium ATP exchange in the absence of proton gradient by the H+-ATPase from yeast plasma membranes.
      ,
      • Goffeau A.
      • de Meis L.
      Effects of phosphate and hydrophobic molecules on two mutations in the β-strand sector of the H+-ATPase from the yeast plasma membrane.
      ).
      Figure thumbnail gr11
      FIGURE 11André Goffeau from the Université Catholique de Louvain in Belgium, well known for his important findings on the H+-ATPase and DNA sequencing.
      Like the Ca2+-ATPase, the (Na+ + K+)-ATPase could be phosphorylated by Pi, and after the addition of excess Na+, the phosphoenzyme could transfer the phosphate to ATP, synthesizing ATP after a single catalytic cycle in the absence of a transmembrane gradient. A few years later, it was found that in the absence of a gradient, a 200-fold decrease in the Pi concentration needed for half-maximal phosphorylation was obtained when 40–60% of the water of the assay medium was replaced with dimethyl sulfoxide. At this solvation concentration, the phosphoenzyme was not able to transfer its phosphate to ADP after the addition of excess Na+. However, synthesis took place if the dimethyl sulfoxide concentration was decreased suddenly, as described for the muscle Ca2+-ATPase. These findings indicated that, as with Ca2+-ATPase, the conversion of the phosphoenzyme from a low- to high-energy form of the (Na+ + K+)-ATPase is related to a hydrophobic-hydrophilic transition at the catalytic site (
      • Post R.L.
      • Toda G.
      • Rogers F.N.
      Phosphorylation by inorganic phosphate of sodium plus potassium ion transport adenosine triphosphatase. Four reactive states.
      ,
      • Jørgensen P.L.
      Mechanism of the Na+,K+ pump. Protein structure and conformations of the pure (Na+ + K+)-ATPase.
      ,
      • de Moraes V.L.
      • de Meis L.
      ATP synthesis by the (Na+ + K+)-ATPase in the absence of an ionic gradient. Effects of organic solvent.
      ).

      Thermogenesis: Heat Production by the Ca2+-ATPase

      Heat generation plays a key role in the regulation of the energy balance of the cell, and alterations of thermogenesis are noted in several diseases such as adiposity and thyroid hormone alterations (
      • Nunes M.T.
      • Bianco A.C.
      • Migala A.
      • Agostini B.
      • Hasselbach W.
      Thyroxine induced transformation in sarcoplasmic reticulum of rabbit soleus and psoas muscles.
      ,
      • Clausen T.
      • Van Hardeveld C.
      • Everts M.E.
      Significance of cation transport in control of energy metabolism and thermogenesis.
      ,
      • Levine J.A.
      • Eberhardt N.L.
      • Jensen M.D.
      Role of nonexercise activity thermogenesis in resistance to fat gain in humans.
      ,
      • Janský L.
      Humoral thermogenesis and its role in maintaining energy balance.
      ,
      • Lowell B.B.
      • Spiegelman B.M.
      Towards a molecular understanding of adaptive thermogenesis.
      ). Skeletal muscle is by far the most abundant tissue of the human body and accounts for >50% of the total oxygen consumption in a resting human and can rise up to 90% during very active muscular work. Different studies have indicated that the hydrolysis of ATP by the muscle Ca2+-ATPase is a major heat source contributing to animal thermogenesis (
      • Clausen T.
      • Van Hardeveld C.
      • Everts M.E.
      Significance of cation transport in control of energy metabolism and thermogenesis.
      ,
      • Janský L.
      Humoral thermogenesis and its role in maintaining energy balance.
      ). During catalysis, a significant part of the energy derived from ATP hydrolysis is dissipated into the surrounding medium as heat. Until recently, it was assumed that the portion converted into heat was always the same, as if the energy released during ATP cleavage was divided into two non-interchangeable parts, one used for work (Ca2+ transport) and the other converted into heat. In more recent reports (
      • de Meis L.
      Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum.
      ,
      • de Meis L.
      Role of the sarcoplasmic reticulum Ca2+-ATPase on heat production and thermogenesis.
      ,
      • de Meis L.
      Uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase. Regulation by ADP.
      ), we found that depending on the conditions used, the amount of heat released during the hydrolysis of ATP by the Ca2+-ATPase can vary between 7 and 32 kcal/mol. This finding indicates that this enzyme is able to handle the energy derived from ATP hydrolysis in such a way as to determine the amount used for Ca2+ transport and the amount that is dissipated as heat. In this view, the total amount of energy released during ATP hydrolysis is always the same, but the fraction of the total energy that is converted into either work or heat seems to be modulated by the Ca2+-ATPase.
      In 1995, Yu and Inesi (Fig. 12) (
      • Yu X.
      • Inesi G.
      Variable stoichiometric efficiency of Ca2+ and Sr2+ transport by the sarcoplasmic reticulum ATPase.
      ) observed that the progressive rise in the Ca2+ concentration in the vesicle lumen promotes the hydrolysis of the phosphoenzyme form 2Ca:E1∼P (Fig. 9B, reaction 7) before being converted into E2-P, and as a result, the cleavage of ATP is processed without a concomitant Ca2+ translocation through the membrane. This finding explained our results of heat production. The rate of reaction 7 determines how much of the energy derived from ATP cleavage is converted into heat and how much into work (Ca2+ translocation). These data were confirmed in different laboratories (
      • de Meis L.
      Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum.
      ,
      • de Meis L.
      Role of the sarcoplasmic reticulum Ca2+-ATPase on heat production and thermogenesis.
      ,
      • de Meis L.
      Uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase. Regulation by ADP.
      ,
      • Yu X.
      • Inesi G.
      Variable stoichiometric efficiency of Ca2+ and Sr2+ transport by the sarcoplasmic reticulum ATPase.
      ,
      • Fortea M.I.
      • Soler F.
      • Fernandez-Belda F.
      Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP.
      ,
      • Logan-Smith M.J.
      • Lockyer P.J.
      • East J.M.
      • Lee A.G.
      Curcumin, a molecule that inhibits the Ca2+-ATPase of sarcoplasmic reticulum but increases the rate of accumulation of Ca2+.
      ,
      • Reis M.
      • Farage M.
      • de Souza A.C.
      • de Meis L.
      Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.
      ,
      • de Meis L.
      • Arruda A.P.
      • Carvalho D.P.
      Role of sarco/endoplasmic reticulum Ca2+-ATPase in thermogenesis.
      ,
      • Grynszpan D.
      • de Meis L.
      The concept of creativity among biochemists.
      ), and reaction 7 was referred to as uncoupled ATPase activity. During transport, a substantial amount of ATP is cleaved through the uncoupled route, and depending on the conditions used, the rate of the uncoupled ATPase activity can be 2–8-fold faster than that the ATPase activity coupled to Ca2+ transport. When the uncoupled ATPase activity is inhibited, there is both an increase in the Ca2+/ATP ratio and a decrease in the yield of heat produced during cleavage of each ATP molecule (ΔHcal), thus abolishing the difference of ΔHcal. Both the coupled and uncoupled ATPase activities have the same Ca2+ dependence. The uncoupled ATPase activity can be specifically inhibited by fluoride and by curcumin, an inhibitor of carcinogenesis. Inhibition of the uncoupled ATPase activity is also observed in the presence of a low ATP and a high ADP concentration and when the assay medium temperature is decreased from 35 °C in homeotherms such as rabbits or below 25 °C in poikilotherms such as fish (
      • de Meis L.
      Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum.
      ,
      • de Meis L.
      Role of the sarcoplasmic reticulum Ca2+-ATPase on heat production and thermogenesis.
      ,
      • de Meis L.
      Uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase. Regulation by ADP.
      ,
      • Yu X.
      • Inesi G.
      Variable stoichiometric efficiency of Ca2+ and Sr2+ transport by the sarcoplasmic reticulum ATPase.
      ,
      • Fortea M.I.
      • Soler F.
      • Fernandez-Belda F.
      Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP.
      ,
      • Logan-Smith M.J.
      • Lockyer P.J.
      • East J.M.
      • Lee A.G.
      Curcumin, a molecule that inhibits the Ca2+-ATPase of sarcoplasmic reticulum but increases the rate of accumulation of Ca2+.
      ,
      • Reis M.
      • Farage M.
      • de Souza A.C.
      • de Meis L.
      Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.
      ,
      • de Meis L.
      • Arruda A.P.
      • Carvalho D.P.
      Role of sarco/endoplasmic reticulum Ca2+-ATPase in thermogenesis.
      ,
      • Grynszpan D.
      • de Meis L.
      The concept of creativity among biochemists.
      ).
      Figure thumbnail gr12
      FIGURE 12Giuseppe Inesi, an expert on Ca2+-ATPase and a dear friend from the University of Maryland School of Medicine in Baltimore.

      Final Comments

      As I mentioned above, I like to spend some of my working time doing research in the social sciences and in science education. In these studies (
      • de Meis L.
      • Longo P.H.
      • Falcáo E.B.M.
      The learning process in science: a study among Brazilian biochemists.
      ,
      • Grynszpan D.
      • de Meis L.
      The concept of creativity among biochemists.
      ), after a significant number of interviews, I found out that both senior and junior scientists are able to identify the competence of the speaker in a lecture by the way he or she presents data. If he or she mentions only the main experiments in simple language that can be easily understood with one set of data clearly related to the next and forming a cycle where beginning and end are obviously interconnected, then he or she is considered to be a top scientist. If a speaker presents various data that are not clearly related and do not form a circular story, then he or she is assumed to have a smaller degree of competence. Finally, if the speaker presents a lot of data, none of it necessarily related to the next, giving the impression that he or she does not clearly know the goal of the lecture, then he or she gets a very low score. Frequently, this type of speaker keeps mentioning that he or she has much more data (and slides) and, should the audience desire it, he or she can go on speaking. Although I am far being sure, I hope that my report was circular, but I know that it takes time to form a circular history. If one has the opportunity to repeat one's tale in different laboratories and in different systems, then one gets more confident, and the feeling is very rewarding. It is not the same temporary, frenetic joy that comes with a new finding. It is a sensation of plentitude that brings reassurance. This, however, does not prevent one from continuing the frantic search for the new. For instance, now I am studying brown adipose tissue in rats. This strange thermogenic tissue, found in both rats and humans, has a peculiar interconnection between mitochondria and reticulum rich in SERCA1, the same pump isoform found in white muscle. Unlike all other mitochondria, we found that SERCA1 is found not only in the reticulum but also inside mitochondria, and mitochondrial thermogenesis is activated by physiological Ca2+ concentrations. The amazing thing is that other mitochondria (liver, for instance) not only produce very little heat but are simply knocked down by the same Ca2+ concentrations that activate brown adipose tissue mitochondria (Ref.
      • de Meis L.
      • Ketzer L.A.
      • da Costa R.M.
      • de Andrade I.R.
      • Benchimol M.
      Fusion of the endoplasmic reticulum and mitochondrial outer membrane in rats brown adipose tissue: activation of thermogenesis by Ca2+.
      and references within).
      I had a strategy that proved useful for my students and me. When I went abroad and visited a colleague hoping to improve my way of thinking, I was able to talk with most of them for only a short period of time due to the many duties of a laboratory leader. Thus, I changed strategies. After several years working together, there is a tendency among the members of a team to think in a similar way. Thus, to expose the whole team to different forms of thinking, we invited scientists to our laboratory. That proved helpful in breaking the vicious thinking circle not only for me but for all the staff members as well. When outside his or her laboratory, the scientist is free of administrative obligations and can spend more time discussing science with others.
      I like to work on two different types of research projects. The first type involves working on my own with my technician without involving students. In this type of work, I like to chase the improbable. These projects frequently result in no publication, a lot of reagent wasted, and intense frustration not shared by collaborators, but if something comes out, then I come a little closer to the ways of nature, and the emotion is also stronger and unshared.
      In the second type of project, I work in strict collaboration with my students, including doing experiments together at the bench. The reasoning is that students cannot dedicate a limitless amount of time to the project. The university imposes strict time periods to graduate. In addition, students need publications to prove what they have done. As I have described above, to be a student is not an easy task. Finally, I must confess that when working with students, it never has been clear to me who learns more: me or the students. Frequently, students tell me that they are insecure and not sure if science is really their vocation. I have learned that a good test is how you feel on Friday and Sunday. If you feel happy on Friday because the next two mornings you do not need go to work and sad on Sunday because tomorrow it will start all over again, then you are in trouble because work is just work, a tedious task that one has to do to survive. Perhaps this is the root of the so-called Friday happy hour. However, if there is no difference in humor between Friday and Sunday, then you do not work; you simply follow your vocation, and you are doing what you really like to do. It is painful for me not be able to go to the laboratory when other matters prevent me from doing so.

      Acknowledgments

      Most of the work described in this Reflections article was supported by the Financiadora de Estudos e Projetos, the Conselho Nacional de Desenvolvimento Científico e Tecnológico, the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

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