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Volume 270, Number 12, Issue of March 24, 1995 pp. 6607-6614
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Some Unique Characteristics of Thylakoid Unisite ATPase (*)

(Received for publication, August 26, 1994; and in revised form, December 27, 1994)

Shiying Zhang (§),  ,  André T. Jagendorf (¶)

From the Plant Biology Section, Cornell University, Ithaca, New York 14853

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Under unisite conditions (ratio of ATP to chloroplast coupling factor (CF(0)CF(1)), approximately 1:2.8), spinach thylakoid ATPase depends on prior reductive activation of CF(1), just as multisite ATPase does, and is sensitive to removal of CF(1) by EDTA. Faster rates in room light than in semidarkness and up to 80% inhibition by uncouplers only in room light indicate a strong effect of proton-motive force, which can be provided by room light. In addition, unisite ATPase is inhibited by azide as long as some ADP is bound to the CF(1).

Several differences were found between unisite and multisite ATPase. 1) The unisite activities of both membrane-bound and free enzyme were stimulated up to 3-fold by 4 mM free MgCl(2) (a strong inhibitor of multisite ATPase). 2) Thylakoid unisite ATPase was inhibited by sulfite (50% inhibition at 5 mM), a powerful activator of multisite ATPase. This inhibition is attributed to a nonspecific ionic strength effect. 3) Unisite ATPase was inhibited by trypsin treatment, which increases multisite ATPase severalfold. 4) The pH profile of thylakoid unisite ATPase is somewhat different from that of multisite. 5) Alkylation of Cys-89 of the subunit by N-ethylmaleimide did not affect the unisite activity, but inhibited multisite activity more than 90%.

INTRODUCTION

Of the three catalytic sites of F-type ATPases, one was found to have higher affinity for ATP than the others(1, 2, 3, 4) . The properties of this site have been studied extensively with both mitochondrial (1, 2, 5, 6) and bacterial F(1)-ATPases(3, 7) . The rate of ATP hydrolysis when only one site is operating is between 10^4 and 10^6 times slower than with multisite operation. There are also some different characteristics, for instance, a lower pH optimum in both MF(1)(^1)(6) and EF(1)(7) .

With isolated F(1)-ATPases studied so far, azide is a strong inhibitor of multisite, but not of unisite ATPase(6, 7, 8, 9, 10) , and is therefore considered to be a specific inhibitor of cooperative interactions between the catalytic sites. Lack of a major anion effect on unisite activity appears to be another common property of MF(1) and EF(1)(11, 12, 13) . However, a number of differences were also found. For instance, millimolar free inorganic phosphate activated MF(1) unisite ATPase 3-fold(14) , but slightly inhibited Escherichia coli unisite ATPase(7, 15) . Studies of chloroplast unisite ATPase, including its kinetic properties, have been reported by Gräber's group(4, 16) . We were interested in exploring further biochemical parameters of thylakoid unisite ATPase, to compare with unisite ATPase in other types of F(1), and with chloroplast multisite ATPase. This has included the effects of different ions, uncouplers, trypsin treatment, pH, energy transfer inhibitors, light, azide, and modification by MalNEt. Our results indicate that chloroplast ATPase shares some properties with other F-type ATPases, but it has some unique characteristics at the unisite level. Also the characteristics of the unisite catalysis differ significantly from those of multisite activity of the same enzyme.

EXPERIMENTAL PROCEDURES

Materials

All reagent grade chemicals, biochemicals, and enzymes were purchased from Sigma, except for [-P]ATP (from NEN-DuPont), Elon (from Eastman Kodak Co.), and ammonium molybdate (from Fluka Chemie AG). Both CF(1) and thylakoids were prepared from spinach leaves bought from local supermarkets.

Preparation of Latent Thylakoids

Thylakoids were prepared as described previously(17) , with choline chloride in the grinding buffer (25 mM Tricine-NaOH, pH 8.0, 300 mM sorbitol, 200 mM choline chloride, 5 mM MgCl(2), 2 mg/ml bovine serum albumin, and 5 mM ascorbate). Thylakoids were washed once and resuspended in the same grinding buffer at a Chl concentration of 1.0 mg/ml. The Chl concentration was determined as described by Wintermans and DeMots (18) .

Reduction of Thylakoids

Thylakoids reduced by DTT in the light were prepared as described elsewhere(17) . In some experiments, reduction was accomplished in the dark by resuspending thylakoids in a buffer containing 20 mM Tricine-NaOH (pH 8.0), 50 mM NaCl, 2 mM MgCl(2), and 20 mM DTT, at a Chl concentration of 0.5 mg/ml. These thylakoids were kept on ice for about 2 h before use.

Preparation of Nucleotide-depleted Thylakoids

Latent thylakoids were isolated as described above. After washing once with the same grinding buffer, the thylakoids were washed twice in a buffer containing 2 mM Tricine-NaOH (pH 8.0), 50 mM NaCl, and 1 mM MgCl(2) and then resuspended in the same buffer at 0.5 mg Chl/ml. After dilution to 0.2 mg of Chl/ml into light-activating buffer (20 mM HEPES, pH 8.0, 50 mM NaCl, 5 mM MgCl(2), 50 µM PMS, and 20 mM DTT), the suspension was illuminated in white light (1.1 mE/m^2bullets) for 3 min at 25 °C. Before turning the light off, 10 volumes of dilution buffer (120 mM KCl, 0.5 mM EDTA, 5 mM Tricine-NaOH, pH 8.0, and 5 mM DTT) were added. The thylakoids were spun down and resuspended in a small volume with the same dilution buffer. After another cycle of illumination-dilution-centrifugation, the thylakoids were resuspended in 20 mM Tricine-NaOH (pH 8.0), 2 mM MgCl(2), 50 mM NaCl, and 10 mM DTT. This protocol is modified from the method of Du and Boyer(19) . Residual nucleotides were measured in control and treated thylakoids by denaturing with perchloric acid, extraction of the perchloric acid with 0.5 M tri-octylamine dissolved in chloroform(20) , then measuring ATP with luciferin and purified luciferase (Sigma). Total adenylates in the samples were measured following conversion of ADP to ATP by incubation with phosphocreatine and creatine phosphokinase.

Removal of CF(1) from Thylakoids by EDTA

Reduced thylakoids were washed once with 10 mM NaCl, 10 mM DTT, 10 mM Tricine-NaOH at pH 7.8, then resuspended to 0.1 mg of Chl/ml in 0.75 mM EDTA, 10 mM DTT, 0.5 mM Tricine-NaOH, pH 7.8, for 15 min on ice, with mixing two to three times during the incubation. After centrifugation, the thylakoids were resuspended in a small volume of 20 mM Tricine-NaOH, pH 8.0, 10 mM DTT, and 50 mM NaCl with the Chl concentration at about 0.5 mg/ml.

Trypsin Digestion of Thylakoids

Reduced thylakoids were incubated on ice, at a final concentration of 500 µg of Chl/ml, with trypsin between 25 and 100 µg/ml (weight ratio of 1/5 to 1/20). The digestion was run for 10 min and stopped by addition of soybean trypsin inhibitor to 10 times the concentration of trypsin. The treated thylakoids were spun down in a microcentrifuge at 4 °C for 5 min and resuspended in 10 mM Tricine-NaOH, pH 8.0, 50 mM NaCl, 1 mM MgCl(2), and 10 mM DTT.

Alkylation of Thylakoid ATPase in the Light

Thylakoids were washed with a buffer containing 20 mM Tricine-NaOH, pH 8.0, 50 mM NaCl, and 2 mM MgCl(2). Before being irradiated, the thylakoids were resuspended in the same buffer plus 50 µM PMS, at a Chl concentration of 0.5 mg/ml. After illumination (1.1 mE/m^2bullets) for 2 min at 25 °C, MalNEt was added to a final concentration of 5 mM. The alkylation was allowed to run under continuing illumination for 2 min. After the light was turned off, 20 mM DTT was added to remove residual free MalNEt. The thylakoids were then spun down, resuspended in the original buffer with 10 mM DTT, and stored on ice in the dark for more than 1 h to reduce the CF(1) completely. The control sample was treated with deionized water instead of MalNEt.

Thylakoid Unisite ATPase Assay

The CF(0)CF(1) content of thylakoids was assumed to be 1.4 nmol/mg of chlorophyll(16) . The assay conditions were established through numerous trial experiments. All reactions were carried out in Eppendorf tubes, at room temperature (22 °C), and either in room light (10-25 µE/m^2bullets) or in dim light (<2 µE/m^2bullets).

The reaction mixture (60 µl) contained 20 mM Tricine-NaOH, pH 8.0, 10 mM DTT, 5 mM MgCl(2), and thylakoids at 30 µg of Chl/ml. Other additions are as indicated under ``Results.'' Thylakoids were first added to the reaction mixture without ATP, then 51 µl of this mixture were added to another Eppendorf tube containing 9 µl of [-P]ATP (100 nM, with specific activity 10^8 cpm/nmol of ATP) to start the reaction, with quick mixing by pipetting. For general activity assays, the reaction was usually run for 60 s before addition of 25 µl of the stop solution, 30 mM Hg(NO(3))(2) in 5 M acetic acid. After centrifuging, 50 µl of the supernatant were assayed for P(i) (see below) released by unisite ATPase. For kinetic assays, 90-µl timed aliquots were removed from a larger volume of reaction medium and placed in an Eppendorf tube containing 40 µl of stop solution.

To determine pH profiles, a solution containing the buffers MES (pK(a) 6.0), MOPS (pK(a) 7.1), Tricine (pK(a) 7.9), and CHES (pK(a) 9.0) was mixed with Mg and DTT prior to adjusting the pH. The final concentration of each buffer was 20 mM, 5 mM Mg, and 10 mM DTT.

Soluble CF(1) Unisite ATPase Assay

Soluble CF(1) was purified according to previous descriptions (21) and stored in 50% saturated ammonium sulfate, 50 mM Tris-Tricine, pH 8.0, 1 mM NaN(3), 1 mM ATP. Before each assay, the precipitate was pelleted and redissolved, then desalted twice through Sephadex G-50 (22) equilibrated with 50 mM Tricine-NaOH, pH 8.0, 0.1 mM EDTA, 20 mM DTT, and 20% glycerol. After addition of 20 mM DTT and 50 mM Tricine-NaOH, pH 8.0, the enzyme solution was incubated at room temperature for 2-3 h, and then 15 min at 37 °C, allowing the enzyme to be completely reduced.

For unisite ATPase, the reaction medium (50 µl) contained 50 mM Tricine-NaOH, pH 8.0, 10 mM DTT, with either 5 mM MgCl(2), or 8 mM CaCl(2). The ratio of ATP to CF(1) was 1:8 with 0.1 µM [-P]ATP and 0.8 µM CF(1) (0.33 mg protein/ml). The reaction was conducted at room temperature for 60 s.

ATPase Assay

The procedure for determination of P(i) released from labeled [-P]ATP was modified from the method of Fromme and Gräber(16) . To each tube containing 50 µl of sample, 200 µl of 4% (w/v) ammonium molybdate in 2.5 M H(2)SO(4), containing 1% (v/v) saturated bromine water were added. Water-saturated butyl acetate (150 µl) was added, and the sample was vortexed to separate complexed P(i) from remaining [-P]ATP. From the organic phase 60 µl were taken to count in a liquid scintillation spectrometer. Background counts, measured in samples prekilled before adding [-P]ATP, were subtracted from each experimental sample. The total counts of [-P]ATP in the samples were measured every day and were used to calculate the specific activity of the labeled ATP.

RESULTS

Unisite Hydrolytic Activity of Thylakoids Is Due to CF(1)

Since extremely low ATPase activities are found under unisite conditions, we needed to be sure that they were due to CF(1)CF(0) and not to some contaminating enzyme. Three kinds of evidence supported the CF(1) origin of observed ATPase.

First, the formation of P(i) by membrane-bound enzyme was found to be dependent on prior reduction of the thylakoids (Fig. 1). ATP hydrolysis by the reduced enzyme was almost linear up to 40 s at a turnover rate of 0.003 s. By comparison, enzyme activity of the nonreduced thylakoids was insignificant, at most 5% of the rate and that for only the first 20 s. Second, treating thylakoids with EDTA at low ionic strength removes CF(1) and causes uncoupling reduced unisite activity more than 90% (data not shown). Third, preliminary experiments showed up to 50% inhibition was found by addition of an antiserum against CF(1) (data not shown), but leq10% inhibition was seen with the preimmune serum. In these experiments the reduced thylakoids and antiserum were incubated at 0 °C for 30 min and centrifuged, and the thylakoids were resuspended and used for unisite ATPase assay. Other conditions would have to be tested to see if greater inhibition might be possible.

Figure 1: Thylakoid unisite ATPase is activated by DTT plus light reduction. The reaction medium (60 µl) contained 50 mM Tricine-NaOH, pH 8.0, 2 mM MgCl(2), 1.8 µg of Chl, and 15 nM [-P]ATP, with (for reduced thylakoids) or without (for control thylakoids) 10 mM DTT. Thylakoids were either untreated (hollow circles) or previously reduced by DTT in the light (filled circles). The reactions were run in room light (20 µE/m^2bullets) at room temperature (22 °C).


Maximal Activity Requires a Proton-Motive Force

We were surprised to find that unisite ATPase of thylakoids was inhibited strongly by uncouplers, including gramicidin (Fig. 2), nigericin, and NH(4)Cl (Table 1). The highest inhibition (87%) was exerted by the combination of 0.5 µM gramicidin and 2 mM NH(4)Cl. Valinomycin, an ionophore for K or ammonium ions, also inhibited the thylakoid unisite ATPase, but less effectively than others (Table 1). These results indicated a major part of unisite ATPase thylakoids requires a proton-motive force. A smaller proportion (about 20%) is active without that help. As expected, soluble CF(1) unisite activity was not affected by uncouplers (data not shown).

Figure 2: Gramicidin inhibits thylakoid unisite ATPase in room light, but not in semidarkness. The reaction mixture (60 µl) contained 10 mM Tricine-NaOH (pH 8.0), 10 mM DTT, 5 mM MgCl(2), 1.8 µg of Chl, and 15 nM [-P]ATP. The reactions were run for 60 s at room temperature, in either room light (20 µE/m^2bullets; hollow circles) or semidarkness (<2 µE/m^2bullets; filled circles).


Since it is known that CF(1) activation occurs with a relatively low pmf(23, 24) , it seemed conceivable that even room light may have generated enough electron transport for this purpose. Accordingly the same experiments were carried out in very dim light (<2 µE/m^2bullets); and indeed, control rates were much lower, and the uncoupler effect disappeared (Fig. 2).

Unisite Activity Is Partially Inhibited by Azide and Venturicidin

Azide inhibits multisite ATPase of mitochondrial F(1), but not unisite ATPase(6) . Surprisingly, azide inhibits thylakoid unisite ATPase, 50% at 1 mM (Fig. 3). Since azide has no effect on photophosphorylation, we cannot ascribe this inhibition to any uncoupling effect. The effective concentration is in the same range, 0.2-2 mM, as reported by others (6, 8, 25) for multisite ATPase and is not another manifestation of the ionic strength effect (see below). Unlike the case for thylakoid multisite ATPase (25) this azide inhibition was not reversed by sulfite (data not shown).

Figure 3: Azide inhibits regular, but not ADP-depleted thylakoid unisite ATPase. The assay conditions were as in Fig. 2, in room light, with control (hollow circles) or ADP-depleted thylakoids (filled circles).


It was shown previously that azide inhibits by enhancing the binding of inhibitory MgADP (26, 27) to one of the three catalytic sites in isolated thylakoids. To obtain ADP-depleted membrane-bound ATPase, thylakoids were diluted in the light and washed extensively with EDTA at high ionic strength right after illumination, as described by others (4, 18) . Measurements of bound adenylates showed a drop from 1.70 down to 0.76 nmol of bound ADP per nmol of CF(1); and a drop of ATP from 1.77 to 0.95 nmol/nmol of CF(1). The depleted thylakoids showed almost no azide inhibition of unisite ATPase (Fig. 3).

In our previous studies, we found that venturicidin can specifically inhibit thylakoid ATPase by blocking proton pumping through CF(0)(17) . Under unisite conditions, a maximum of 30-40% inhibition was observed in a number of experiments.

Anions Inhibit Unisite Activity of Thylakoid ATPase

Much to our surprise, sulfite, which greatly stimulates steady state ATP hydrolysis by reduced thylakoids(29) , inhibited unisite ATP hydrolysis by thylakoids (Fig. 4A), up to 84% at 50 mM. To determine whether the inhibition was a side effect, due perhaps to high salt or osmotic strength, we titrated sulfite and sorbitol side by side, along with some other salts (Fig. 4A). It is evident that sulfite inhibition of the unisite hydrolytic activity is shared by other salts and is not unique. Since anions with higher negative charges (sulfite, sulfate) have a stronger effect than those with a single charge (chloride), the inhibition is due to the anions rather than to a general ionic strength effect. For instance, 50% inhibition was obtained with 5 mM sodium sulfite, but 40 mMNaCl was required (Fig. 4A). Unlike multisite ATPase, the unisite ATPase responses to sulfite and sulfate are indistinguishable. As these were all salts with Na as the cation, it strengthens the idea that the anions are inhibitory. The failure of sorbitol to inhibit even at high concentrations shows that this is not an osmotic strength effect.

Figure 4: A, salts inhibit thylakoid unisite ATPase. Assay conditions as in Fig. 2, in room light. The reactions were run for 60 s. B, sulfite stimulates soluble CF(1) unisite ATPase, but other salts have no effect. The reaction mixture (60 µl) contained 50 mM Tricine-NaOH (pH 8.0), 10 mM DTT, 5 mM MgCl(2), 100 nM [-P]ATP, 800 nM reduced CF(1), and different salts at different concentrations, as indicated. The reactions were run for 60 s at room temperature.


We conducted similar experiments with purified soluble CF(1) (Fig. 4B). Only sulfite had an effect on the soluble enzyme activity, all other salts had no effect. As expected sulfite stimulated the soluble unisite activity, opposite to its effect on the thylakoid bound enzyme.

The inhibitory effect of anions on thylakoid unisite ATPase can be observed only when the reaction medium contains mM levels of free Mg (Fig. 5) and the assays are conducted in room light. Without Mg in the assay medium, or if the assay is performed in very dim light (<2 µE/m^2bullets), the rates are much lower (see below), and added NaCl or Na(2)SO(3) have either no effect or are slightly stimulatory.

Figure 5: Thylakoid unisite ATPase is inhibited by salt only in the presence of Mg. The assay conditions were similar to those in Fig. 2, in room light, except that these assays were performed in either the presence (hollow symbols) or absence (filled symbols) of 5 mM MgCl(2). Open squares and filled triangles, NaHSO(3); open triangles and filled circles, NaCl.


Inorganic phosphate stimulates soluble MF(1)(14) , but inhibits EF(1) unisite ATPase(6, 15) . With thylakoid unisite ATPase, less than 1 mM phosphate enhanced the unisite ATPase activity, but inhibited at concentrations above 1 mM (not shown). However, the stimulatory effect of phosphate was found to be due to the formation of ATP under room light (10-20 µE/m^2bullets), presumably from the tightly bound ADP, thereby raising the net ATP concentration. The inhibitory effect of phosphate is probably due to the same anion inhibition found with other salts. With soluble CF(1), a slight stimulatory effect was also observed under unisite conditions, but much less than that of sulfite (data not shown).

Maximal Unisite Activity Requires Free Metal Ions

When inhibition by added salts was found, as in Fig. 4and other experiments, 5 mM MgCl(2) was already present. Because of the unexpected anion inhibition of unisite catalysis of thylakoid ATPase, it was of interest to test for effects of cations. In a medium virtually lacking salts, the unisite activity was very low. Increasing concentrations of Mg, Ca, and Na salts up to 5 mM stimulated the low hydrolytic activity to some extent (Fig. 6A). The extent of stimulation varied depending on the cation used, with Mg being the most effective. For instance, 5 mM Mg stimulated the enzyme activity more than 4-fold, whereas only about 3- and 2-fold stimulations were observed with Ca or Na at the same concentrations (NaCl stimulation was observed only when there was no MgCl(2) in the reaction medium). At higher concentrations, however, all the salts showed some inhibitory effect, probably due to the anion inhibition becoming dominant.

Figure 6: A, cation stimulation of thylakoid unisite ATPase. The reaction mixture was similar to that in Fig. 2, in room light, except for the presence of different salts (as indicated) at the concentrations indicated. B, cation stimulation of soluble CF(1) unisite ATPase. The conditions were similar to those in Fig. 2, with additions of CaCl(2) or MgCl(2) as indicated.


More surprisingly, a similar cation effect was also found with soluble CF(1) unisite ATPase (Fig. 6B), except that here Ca was more effective than Mg, in contrast to the thylakoid-bound enzyme. For instance, Mg was optimal at 5 mM, giving a 2-fold stimulation; but with Ca, a 7-fold stimulation was observed at 10 mM.

Trypsin Treatment Inhibits Unisite Activity

Treating thylakoid membranes with trypsin led to higher multisite ATPase activity(30) , but nothing was known about the effect of this treatment on unisite ATPase. We found that trypsin only inhibits unisite ATPase, ranging from 24% inhibition with a ratio of trypsin to chlorophyll of 1:20, to 76% inhibition at a ratio of 1:5 (data not shown).

We could think of two possibilities to explain this result. The first is that trypsin digestion might destroy the high affinity site, as the result of cleaving the alpha and/or beta subunits(30, 31) . If so, soluble CF(1) should also have been inhibited. However, trypsin digestion increased soluble unisite activity only to some extent (data not shown). Thus it is not likely that inhibition of thylakoid unisite ATPase by trypsin is due to a loss of one or the other of the large subunits. The second possibility, which is accordingly more likely, is that the slight uncoupling effect of trypsin treatment (30) might cause the inhibition.

The pH Profile of Unisite ATPase Is Different from That of Multisite

For MF(1)(6) and E. coli F(1)(7) , the pH profiles of unisite catalysis were found to be completely different from that of multisite. However, the pH response of thylakoid unisite ATPase (Fig. 7) was very close to that of multisite, showing sensitivity to both high (pH >9.0) and low pH (pH <7.5). But some differences still exist. These include the optimal pH, which is 8.0 for unisite and 8.5 for multisite; and inhibitions at a high pH, where unisite is more sensitive than multisite.

Figure 7: The pH profiles of thylakoid ATPases. The unisite (hollow circles) reaction medium (60 µl) contained 20 mM MES, 20 mM MOPS, 20 mM Tricine, 20 mM CHES, 5 mM MgCl(2), 10 mM DTT, 15 nM [-P]ATP, and 1.8 µg of Chl. Multisite (filled circles) ATPase was run with 5 mM ATP, and other conditions were the same. The medium pH was adjusted prior to the addition of ATP and thylakoids. The rates are shown relative to each respective maximal rate. These were 0.002 mol of P(i)/mol of CF(0)CF(1) for unisite hydrolysis, and 28.5 for multisite.


Interestingly, very similar pH profiles were also observed with soluble CF(1) ATPase (data not shown). Thus the differences between thylakoid unisite ATPase and other F-type ATPases in the pH profiles are probably not due to the thylakoid membranes, but rather to the reactivity of CF(1) itself.

Sulfite and Trypsin Treatment Stimulate Unisite ATPase in the Dark

In dim light (<2 µE/m^2bullets) sulfite actually stimulated unisite ATPase about 2-fold at 10 mM (Fig. 8), unlike its effect in room light. This was the same with or without uncoupler present (data not shown). However, at higher concentrations (>10 mM), sulfite does not stimulate dim light ATPase, rather slightly inhibits it.

Figure 8: Sulfite stimulates thylakoid unisite ATPase in semidarkness. The conditions were as in Fig. 2, in room light (hollow triangles) or semidarkness (closed circles). A, reduced thylakoids; B, thylakoids reduced and treated with trypsin.


Similar results were also obtained with trypsin-treated thylakoids. In dim light, sulfite stimulated the trypsin-cleaved thylakoid unisite ATPase activity (Fig. 8), with an optimal concentration at 20 mM, increasing the rate 3-fold. However, further increasing sulfite concentrations again resulted in some inhibition.

MalNEt Alkylation of Cys-89 of the Subunit Has No Effect on Unisite Activity

Cys-89 is not accessible in the inactive form of thylakoid ATPase, but becomes accessible upon membrane energization (32) . Both Nalin et al.(32) and Cohen (33) showed that modification of this cysteine residue by MalNEt inhibits both ATP synthase and ATPase activities (multisite). More recently, however, it was found that alkylation of Cys-89 stimulates the rates of nucleotide exchange. (^2)To test the effect of this modification on unisite activity, we treated thylakoids with MalNEt in the light without DTT reduction (to ensure the modification did not occur on the SH groups of the reduced disulfide bond). ATPase of the treated thylakoids was assayed under both unisite and multisite conditions (Table 2). Alkylation of Cys-89 by MalNEt inhibited the unisite ATPase only 10%, but inhibited the multisite activity 90%. This suggests a function of Cys-89 in modulating the interactions between different catalytic sites.

DISCUSSION

Data presented in this report demonstrate differences between the unisite and multisite activities of thylakoid ATPase and between chloroplast F(1) and mitochondrial or E. coli F(1)- ATPases. These results could be useful for further understanding the molecular mechanism of the enzyme.

First, we wanted to make sure that the very low levels of ATP hydrolysis are due to CF(0)CF(1). The dependence of unisite activity on prior reduction of the thylakoids by DTT in the light, loss of activity when thylakoids are depleted of CF(1) by EDTA treatment at low ionic strength, and partial inhibition due to an antiserum against CF(1) are all characteristics shared by normal thylakoid multisite ATPase. It is highly improbable that another enzyme could be responsive to these same inputs.

The rates of unisite ATPase we observed in room light were comparable to those obtained in a similar study by Fromme and Gräber(16) , despite the differences in experimental conditions. They used either light or an acid/base jump to reactivate the reduced thylakoid enzyme before each assay, without comment on any activity in the absence of this activation. They also used a high concentration of uncoupler (3-6 mM NH(4)Cl) in the assay medium after reactivating the enzyme(16, 34) .

Our finding of inhibition of unisite catalysis by uncouplers in room light but not in dim light indicates a requirement for a small proton-motive force for all but a small fraction of the unisite activity (Fig. 2, Table 1). This could not have been due to carryover of some pmf from the previous reductive activation, especially because uncoupler inhibition was found to the same extent when the thylakoids had been reduced by incubating with DTT in the dark, without PMS. Generation of a pmf under room light (<25 µE/m^2bullets) and with no added electron-carrying dye initially seemed unlikely. However, a very low pH gradient, perhaps only 2.0 units, is sufficient to activate CF(1)(23, 24) . With either 9-aminoacridine or neutral red as indicators, we did observe a thylakoid pmf forming due to these low light intensities and without added redox dyes. (^3)The pattern of electron flow, in the absence of added redox dyes, that can operate at this low light level has not been defined. Preliminary experiments with inhibitors (dichlorophenyl dimethylurea for PSII; high levels of KCN for plastocyanin and PSI) seem to indicate involvement of only PSI; but further work is needed for a better definition.

It was surprising to see that thylakoid unisite ATPase (Fig. 3) and that of soluble CF(1) are partially inhibited by azide (Fig. 3). Both MF(1) and EF(1) unisite activities, and even that of soluble CF(1) Ca-dependent ATPase (9) , are not inhibited by azide(6, 10) . The azide inhibition of thylakoids (26, 35) as with other systems, is considered to be due to tightening the binding of inhibitory MgADP to one of the adenylate binding sites. Thereby it interferes with site-site interactions and causes inhibition of steady state ATP hydrolysis. This is likely to be the case with thylakoid unisite activity also, since azide did not inhibit thylakoids which had been partially depleted of bound ADP. Either the unisite binding and hydrolysis occurs at the site of tight ADP binding, or ATP hydrolysis at only one site is still subject to allosteric control by the tightly bound ADP. Either way, any means of loosening or releasing the bound ADP would stimulate unisite activity.

As observed with MF(1)(28) , venturicidin only partially inhibits thylakoid unisite ATPase. Matsuno-Yagi and Hatefi (28) proposed that venturicidin binding to the F(0) sector freezes its structure, but does not block the proton channel. Since under unisite conditions only one set of protons is expected to move through any one channel, a slow translocation rate would be sufficient to support the very slow ATP hydrolysis.

The effects of salts on thylakoid unisite catalysis include stimulation at low concentrations (0-5 mM) and inhibition at higher levels. The inhibition is a nonspecific anion effect (Fig. 4A) and is not found with either unisite activity of soluble CF(1) (Fig. 4B) or thylakoid multsite ATPase. Since it does not occur in dim light, the salts may be interfering with either the unknown electron flow path or in some other way with generation of a pmf. While the cause is not known, the phenomenon explains why the usually strong potentiation by sulfite cannot be observed at these substrate levels.

Part of the stimulation by low levels of salts may represent a modification of thylakoid structure. While the divalent cations Ca and Mg are most effective, even Na (Fig. 6A) and Tricine (data not shown) have some effect. It was found much earlier (36, 37) that at very low salt concentrations the grana structure of thylakoids disappears; but it can be restored by adding salts, especially of divalent cations.

However, the greater effectiveness of Mg, and especially the strong stimulation of soluble CF(1) unisite ATPase by divalent cations, indicates a specific role in either catalysis or enzyme structure. This was quite unexpected; the usual concept is that the role of divalent cations is only to complex with the adenylate, forming the true substrate. In the present case the required Mg concentration of 4 to 5 mM is over 5 orders of magnitude higher than the substrate concentration (0.015 µM). It is even more strange in that free Mg is a mild inhibitor of thylakoid multisite ATPase, but a severe inhibitor for soluble CF(1), with a K(i) of 10 µM(38, 39, 40) .

Guerrero et al.(41) proposed that free Mg inhibits ADP release from the tight binding site, thereby stabilizing an inactive form of CF(1). However, its failure to inhibit the unisite activity seems at odds with that interpretation. It seems likely that, on binding to CF(1), Mg induces a conformational change in favor of unisite binding and hydrolysis of the substrate. A similar result was seen in MF(1)(42) where Mg stimulated both unisite catalysis and the rates of ADP and P(i) release from the soluble enzyme.

Alkylation of Cys-89 in the light had little effect on unisite, but knocked out most of the multisite activity (Table 2), suggesting another difference between the two types of catalysis. Alkylation of Cys-89 may abolish site-site interactions(43) .

The minor component of unisite activity found in very dim light was not inhibited by salts and (probably for that technical reason) could show some stimulation by sulfite. Sulfite also stimulates rather strongly the unisite activity of soluble CF(1). In both cases, its action presumably has to do with permitting release of the tightly bound, inhibitory MgADP, as in multisite ATPase(41, 44) . Thus once again, with CF(1) some site-site interactions, or at the least an allosteric effect of ADP at an alternative site, are found under unisite catalytic conditions.

Trypsin inhibition of thylakoid unisite ATPase contrasts with the known stimulation of multisite ATPase and also with its stimulation of the unisite activity by soluble CF(1) (data not shown). This inhibition is therefore most probably attributed to its weak uncoupling of thylakoids(30, 31) , since unisite catalysis cannot renew the pmf during the reaction.

In conclusion, thylakoid unisite ATPase can be assayed under room light. Based on so many differences from multisite ATPase, it seems that the high affinity site is either a unique catalytic site or is one of the usual ones with very different characteristics, because of the very low ATP levels.

FOOTNOTES

*
This work was supported in part by National Science Foundation Grant DCB-91-11751 (to A. T. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Molecular Pathophysiology Branch NIDDK, NIH, Building 10, Room 8C-101, 10 Center Dr., MSC 1752, Bethesda, MD 20892-1752.

To whom correspondence should be addressed: Plant Biology Section, Plant Science Bldg., Cornell University, Ithaca, NY 14853. Tel.: 607-255-8940; Fax: 607-255-5407.

(^1)
The abbreviations used are: MF(1), mitochondrial F(1); CF, chloroplast coupling factor; CHES, 2-(cyclohexylamino)ethanesulfonic acid; Chl, chlorophyll; DTT, dithiothreitol; EF(1), E. coli F(1); MalNEt, N-ethylmaleimide; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; pmf, proton-motive force; PMS, N-methyl phenazonium methosulfate; PSI, photosystem I; PSII, photoystem II; Tricine, N-tris(hydroxymethyl)methylglycine; µE, microeinstein.

(^2)
R. E. McCarty, personal communication.

(^3)
W. R. Zipfel and S. Zhang, unpublished results.

ACKNOWLEDGEMENTS

We are grateful for helpful discussions with W. R. Zipfel, R. E. McCarty, and P. Hinkle.

REFERENCES

  1. Grubmeyer, C., Cross, R., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100 [Free Full Text]
  2. Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105 [Free Full Text]
  3. Al-Shawi, M. K., and Senior, A. E. (1988) J. Biol. Chem. 263, 19640-19648 [Abstract/Free Full Text]
  4. Fromme, P., and Gräber, P. (1990) Biochim. Biophys. Acta 1016, 29-42 [Medline] [Order article via Infotrieve]
  5. Cunningham, D., and Cross, R. L. (1988) J. Biol. Chem. 263, 18850-18856 [Abstract/Free Full Text]
  6. Harris, D. A. (1989) Biochim. Biophys. Acta 974, 156-162 [Medline] [Order article via Infotrieve]
  7. Al-Shawi, M. K., and Senior, A. E. (1992) Biochemistry 31, 878-885 [CrossRef][Medline] [Order article via Infotrieve]
  8. Harris, D. A., Boork, J., and Baltscheffsky, M. (1985) Biochemistry 24, 3876-3883 [CrossRef]
  9. Androlajc, P. J., and Harris, D. A. (1990) Biochim. Biophys. Acta 1016, 55-62 [Medline] [Order article via Infotrieve]
  10. Noumi, T., Maeda, M., and Futai, M. (1987) FEBS Lett. 213, 381-384 [CrossRef][Medline] [Order article via Infotrieve]
  11. Kasho, V. N., and Boyer, P. D. (1984) J. Bioenerg. Biomembr. 16, 407-419 [CrossRef][Medline] [Order article via Infotrieve]
  12. Roveri, O. A., and Calcaterra, N. B. (1985) FEBS Lett. 192, 123-127 [CrossRef][Medline] [Order article via Infotrieve]
  13. Wong, S-Y., Matsuno-Yagi, A., and Hatefi, Y. (1984) Biochemistry 23, 5004-5009 [CrossRef][Medline] [Order article via Infotrieve]
  14. Penefsky, H. S., and Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase): Structure, Function, Bioenergenesis of the F F Complex of Coupling Membranes (Papa, S., Altendorf, K., Ernster, L., and Packer, L., eds) pp. 195-204, ISCU Press, Adriatica Editrice, Bari
  15. Muneyuki, E., Yoshida, M., Bullough, D. A., and Allison, W. S. (1991) Biochim. Biophys. Acta 1058, 304-311 [Medline] [Order article via Infotrieve]
  16. Fromme, P., and Gräber, P. (1990) Biochim. Biophys. Acta 1020, 187-194 [CrossRef]
  17. Zhang, S., and Jagendorf, A. T. (1993) Plant Physiol. 101, 127-130 [Abstract]
  18. Wintermans, J. F. G. M., and De Mots, A. (1965) Biochim. Biophys. Acta 109, 448-453 [Medline] [Order article via Infotrieve]
  19. Du, Z., and Boyer, P. D. (1990) Biochemistry 29, 402-407 [CrossRef][Medline] [Order article via Infotrieve]
  20. Khym, J. X. (1975) Clin. Chem. 21, 1245-1252 [Abstract]
  21. Jagendorf, A. T. (1982) in Methods in Chloroplast Molecular Biology (Edelman, M., Hallick, R. B., and Chua, N.-H., eds) pp. 881-898, Elsevier/North Holland Press, Amsterdam
  22. Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899 [Abstract/Free Full Text]
  23. Quick, W. P., and Mills, J. D. (1986) Biochim. Biophys. Acta 851, 166-172 [CrossRef]
  24. Kramer, D. M., Wise, R. R., Frederick, J. R., Alm, D. M., Hesketh, J. D., Ort, D. R., and Crofts, A. R. (1990) Photosynth. Res. 26, 213-222 [CrossRef]
  25. Wei, J-M., Howlett, B., and Jagendorf, A. T. (1988) Biochim. Biophys. Acta 934, 72-79 [CrossRef]
  26. Murataliev, M. B., Milgrom, Y. M., and Boyer, P. D. (1991) Biochemistry 30, 8305-8310 [CrossRef][Medline] [Order article via Infotrieve]
  27. Minkov, I. B., and Strotmann, H. (1989) Biochim. Biophys. Acta 973, 7-12
  28. Matsuno-Yagi, A., and Hatefi, Y. (1993) J. Biol. Chem. 268, 1539-1545 [Abstract/Free Full Text]
  29. Larson, E. M., and Jagendorf, A. T. (1989) Biochim. Biophys. Acta 973, 67-77
  30. Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5915-5920 [Abstract/Free Full Text]
  31. Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914 [Abstract/Free Full Text]
  32. Nalin, C. M., Beliveau, R., and McCarty, R. E. (1983) J. Biol. Chem. 258, 3376-3381 [Abstract/Free Full Text]
  33. Cohen, W. S. (1989) Plant Physiol. 91, 1107-1111 [Abstract/Free Full Text]
  34. Labahn, A., and Graber, P. (1993) Biochim. Biophys. Acta 1141, 288-296 [CrossRef]
  35. Murataliev, M. B., and Boyer, P. D. (1992) Eur. J. Biochem. 209, 681-687 [Medline] [Order article via Infotrieve]
  36. Izawa, S., and Good, N. (1966a) Plant Physiol. 41, 533-543 [Abstract/Free Full Text]
  37. Izawa, S., and Good, N. (1966b) Plant Physiol. 41, 544-552 [Abstract/Free Full Text]
  38. Vambutas, V. K., and Racker, E. (1965) J. Biol. Chem. 240, 2660-2667 [Free Full Text]
  39. Hochman, Y., Lanir, A., and Carmeli, C. (1976) FEBS Lett. 61, 255-259 [CrossRef][Medline] [Order article via Infotrieve]
  40. Posorske, L., and Jagendorf, A. T. (1976) Arch. Biochem. Biophys. 177, 276-283 [CrossRef][Medline] [Order article via Infotrieve]
  41. Guerrero, K. J., Xue, Z., and Boyer, P. D. (1990) J. Biol. Chem. 265, 16280-16287 [Abstract/Free Full Text]
  42. Milgrom, Y. M., and Murataliev, M. B. (1987) FEBS Lett. 222, 32-36 [CrossRef][Medline] [Order article via Infotrieve]
  43. Soteropoulos, P., Ong, A. M. and McCarty, R. I. (1994) J. Biol. Chem. 269, 19810-19816 [Abstract/Free Full Text]
  44. Larson, E. M., Umbach, A., and Jagendorf, A. T. (1989) Biochim. Biophys. Acta 973, 78-85
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