Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M108035200 on October 8, 2001

J. Biol. Chem., Vol. 277, Issue 1, 127-134, January 4, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/1/127    most recent
M108035200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Minic, Z.
Right arrow Articles by Hervé, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Minic, Z.
Right arrow Articles by Hervé, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Catabolism of Pyrimidine Nucleotides in the Deep-sea Tube Worm Riftia pachyptila*

Zoran MinicDagger , Styliani Pastra-Landis§, Françoise Gaill, and Guy HervéDagger ||

From the Dagger  Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, UMR 7631, CNRS, Université Pierre et Marie Curie, 96 Boulevard Raspail F-75006 Paris, France, the § Department of Chemistry, Wheaton College, Norton, Massachusetts 02766-0930, and  Laboratoire de Biologie Marine, UMR 7622, CNRS, Université Pierre et Marie Curie, 7 Quai Saint Bernard, F-75252 Paris, France

Received for publication, August 21, 2001, and in revised form, October 5, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study describes the distribution and properties of enzymes of the catabolic pathway of pyrimidine nucleotides in Riftia pachyptila, a tubeworm living around deep-sea hydrothermal vents and known to be involved in a highly specialized symbiotic association with a bacterium. The catabolic enzymes, 5'-nucleotidase, uridine phosphorylase, and uracil reductase, are present in all tissues of the worm, whereas none of these enzymatic activities were found in the symbiotic bacteria. The 5'-nucleotidase activity was particularly high in the trophosome, the symbiont-harboring tissue. These results suggest that the production of nucleosides in the trophosome may represent an alternative source of carbon and nitrogen for R. pachyptila, because these nucleosides can be delivered to other parts of the worm. This process would complement the source of carbon and nitrogen from organic metabolites provided by the bacterial assimilatory pathways. The localization of the enzymes participating in catabolism, 5'-nucleotidase and uridine phosphorylase, and of the enzymes involved in the biosynthesis of pyrimidine nucleotides, aspartate transcarbamylase and dihydroorotase, shows a non-homogeneous distribution of these enzymes in the trophosome. The catabolic enzymes 5'-nucleotidase and uridine phosphorylase activities increase from the center of the trophosome to its periphery. In contrast, the anabolic enzymes aspartate transcarbamylase and dihydroorotase activities decrease from the center toward the periphery of the trophosome. We propose a general scheme of anatomical and physiological organization of the metabolic pathways of the pyrimidine nucleotides in R. pachyptila and its bacterial endosymbiont.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Riftia pachyptila is a tubeworm living in the close vicinity of deep-sea hydrothermal vents in the Pacific ocean (1). This organism thrives in a community that is almost completely isolated from the biosystems of the rest of the planet. In the vent environment these animals encounter both physical and chemical obstacles, such as elevated pressure, high temperature, and chemical toxicity. R. pachyptila is one of the dominant organisms adapted to this extreme environment, and its anatomical organization is shown in Fig. 1 (2, 3).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1.   The anatomical organization of R. pachyptila. Major organs and tissues assayed for the presence of enzymatic activities. Inset, dissected zones of trophosome tissues (see "Experimental Procedures."

The only tissue of the worm that is in direct contact with the surrounding water is the plume; this plume presents a large, highly vascularized surface, which allows an efficient exchange of metabolites and waste products between the environment and the animal. The other tissues are sequestered within the Riftia tube. The vestimentum is a muscle that the animal uses to position itself in the tube. The trophosome is located within the large sac made by the body wall and terminated by the opisthosome, and it is bathed by coelomic fluid (1).

The trophosome tissue is densely colonized by a sulfur-oxidizing chemoautotrophic endosymbiotic bacterium (4-6). The bacteria are estimated to represent between 15 and 35% of the total volume of the trophosome (4, 7). Most of the metabolite exchanges between the trophosome and the environment are mediated via the vascular system, whose obvious function is to supply host tissues with oxygen and to transport CO2, O2, H2S, NH3, and minerals to the bacteria. The transport of O2 and H2S is ensured by a high molecular weight hemoglobin (8). In return, the bacteria produce metabolic energy from the oxidation of H2S and provide organic compounds as sources of carbon and nitrogen to the worm. In this manner, the nutritional organization involves specific metabolic exchanges between the two organisms.

We reported recently (9, 10) that the first three enzymes of the "de novo " pathway of pyrimidine nucleotides biosynthesis, carbamyl-phosphate synthetase, aspartate transcarbamylase (ATCase),1 and dihydroorotase (DHOase), are present only in the trophosome, and we demonstrated the bacterial origin of these enzymes. In contrast, the succeeding enzymes of de novo pathway, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, are present in all the body parts of the worm as well as in the bacteria. Furthermore, analysis of the enzymes of the "salvage" pathway (cytidine deaminase, cytidine kinase, and uracil phosphoribosyltransferase) in the trophosome strongly suggested that they belong to the worm. Accordingly, none of these enzymatic activities were found in the isolated bacteria. These results indicate that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines. The presence of the enzymes of the salvage pathway in all worm tissues suggests that the synthesis of pyrimidines is indeed possible in those tissues that lack bacteria by starting from intermediary metabolites provided by the trophosome or from the degradation of nucleic acids.

Thus, the presence of the enzymes involved in the salvage pathway in worm tissues implies the presence of their substrates, nucleosides. Furthermore, these nucleosides might provide substrates not only for this pathway but also for the enzymes involved in their "catabolism" (11). In this case the catabolism of nucleosides might represent a possible source of carbon and nitrogen for the worm.

To study catabolism of the pyrimidine nucleotides in R. pachyptila, we investigated the presence of 5'-nucleotidase, uridine phosphorylase, and dihydropyrimidine dehydrogenase (uracil reductase named uracilRase) in the different parts of the worm. 5'-Nucleotidase controls intracellular levels of nucleoside 5'-monophosphate by catalyzing the hydrolysis of the phosphate esterified on the 5'-carbon of the ribose and deoxyribose portions of nucleotide molecules (12). This is a key enzyme for both the salvage pathway and for catabolism because its enzymatic products, nucleosides, can be used in both pathways. Uridine phosphorylase (UPase) catalyzes phosphorolysis of uridine, resulting in the formation of uracil and ribose 1-phosphate (13). Dihydropyrimidine dehydrogenase catalyzes the further reduction of uracil to 5,6-dihydrouracil using NADPH as the reductant (14). After several enzymatic steps, 5,6-dihydrouracil is transformed into CO2, NH3, and malonyl-CoA (15) which can be further metabolized.

The results obtained here emphasize the possibility that pyrimidine catabolism can constitute a source of carbon and nitrogen for the worm. These results together with the data reported previously (9, 10) on de novo and salvage pathways lead us to propose a general organization for the pyrimidine metabolic pathways in R. pachyptila.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Chemicals were obtained from Sigma. The radioactively labeled substrates, L-[14C]aspartate (224.8 mCi/mmol), [2-14C]CMP (60.4 mCi/mmol), and [2-14C]UMP (46.1 mCi/mmol), were also purchased from Sigma. [14C]CTP (450 mCi/mmol) was purchased from the Commissariat à l'Energie Atomique (Saclay, France).

Source and Storage of R. pachyptila Samples-- Samples of the R. pachyptila were collected in the east Pacific volcanic range at a depth of 2600 m (Hope 99 cruise), using the submersible "Nautile," and recovered in a seawater box for the trip to the surface. To preserve tissue identity and avoid interference with subsequent enzymatic tests, the specimens were immediately bled and dissected on board, and isolated organs were frozen in liquid nitrogen, as described previously (9, 10).

Purification of Bacterial Symbiont-- Immediately after collecting and bleeding the animal, the bacterial symbiont was purified by the method proposed by Distel and Felbeck (16), under the conditions described previously (9, 10).

Dissection of Parts of Trophosome Tissue-- To test enzymatic activities in various zones of the trophosome, the frozen trophosome was dissected into central, middle, and exterior parts, each about 3 mm wide. These fractions were treated as described below for the preparation of the cell-free extracts.

Preparation of Protein Extract from Each Organ of R. pachyptila-- Protein extracts from all organs were freshly prepared before the enzyme assays. Frozen tissue (~2 g) was suspended in 6 ml of ice-cold extraction buffer (30 mM Tris-HCl, pH 7.8, 10 mM NaCl, 10 mM KCl, 1 mM L-dithiothreitol, 5% (v/v) glycerol, 30% (v/v) ethylene glycol, 4 µM sodium cacodylate, and the following protease inhibitors: 30 µg/ml phenylmethylsulfonyl fluoride, 0.3 mg/ml EDTA, 0.7 µg/ml pepstatin A, and 0.5 µg/ml leupeptin). 0.1% Triton X-100 was added to the extraction buffer for 5'-nucleotidase activity. The mixture was homogenized in a Potter homogenizer (Teflon pestle). The homogenate was disrupted further by sonication three times for 60 s each with a Biosonik III sonicator at 20 kilocycles/s. The homogenate was then centrifuged at 9000 × g for 20 min, and the resulting dialyzed supernatant was utilized for subsequent enzymatic assays.

5'-Nucleotidase Assay-- Pyrimidine 5'-nucleotidase activity was assayed by following the conversion of [2-14C]UMP to [2-14C]uridine and [2-14C]CMP to [2-14C]cytidine, using a protocol similar to those of West and Beutler (17) and Ellims et al. (18). The standard reaction mixture consisted of 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.5 mM [2-14C]UMP (0.45 mCi/mmol), or 0.5 mM [2-14C]CMP (0.3 mCi/mmol) and 50 µl of tissue extract (0.3-1.0 mg of protein) in a final volume of 150 µl. After incubation at 37 °C for 30 min, the reaction for UMP-5'-nucleotidase was terminated by heating at 95 °C for 2 min, and the incubation mixture was clarified by centrifugation at 10,000 × g for 10 min. The corresponding control was stopped at time 0. Aliquots of 50 µl of the reaction mixture were spotted on DE81 paper squares and dried at room temperature. The paper was transferred to a scintillation vial, and 2 ml of distilled water was added. The vial was then agitated in a shaker for 1 h; the paper was removed, and 8 ml of scintillant was added.

After incubation of the reaction mixture for the assay of CMP-5'-nucleotidase, the reaction was stopped by adding 0.3 ml of 0.15 M Ba(OH)2, 0.3 ml of 0.15 M ZnSO4, and 750 µl of water. Controls were stopped at time 0. The activity for CMP-5'-nucleotidase was determined as described in the protocol of West and Beutler (17).

Substrate specificity studies for 5'-nucleotidase were performed as described in the protocol by Niedzwiecka and Jaroszewicz (19).

Uridine Phosphorylase Assay-- The UPase assay (20) was performed by spectrophotometric measurement. The reaction mixture (200 µl) contained 100 mM potassium phosphate buffer, pH 7.9, 5 mM L-dithiothreitol, 10 mM uridine, and protein extract (0.3-1.0 mg of protein). After incubation at 37 °C for 30 min, the reaction was stopped by the addition of 1.8 ml of 0.01 M NaOH. The absorbance at 290 nm was determined and corrected for a blank that had been stopped at zero time. Specific activity was calculated from the change of absorbance during the first 30 min, using an extinction coefficient for uracil of 5.7 mM-1 cm-1.

Uracil Reductase Assay-- Enzyme activity for this reaction was determined at 37 °C by monitoring the decrease in absorbance at 340 nm that accompanied the conversion of NADPH to NADP+ (21). The reaction mixture contained 50 mM Tris-HCl, pH 7.2, 2.5 mM MgCl2, 2.5 mM 2-mercaptoethanol, 200 µM NADPH, 250 µM uracil, and protein extract (0.5-1.5 mg of protein) in a final volume of 2 ml. The reaction was initiated with uracil and run against a blank containing the identical reaction mixture without uracil. The extinction coefficient used for the oxidation of NADPH to NADP+ was 6.22 mM-1 cm-1.

Aspartate Transcarbamylase Assay-- The aspartate transcarbamylase activity was measured by the radioactive method (9, 10). The standard conditions were 50 mM Tris-HCl, pH 8.0, 20 mM L-[14C]aspartate (0.3 mCi/mmol), and 10 mM carbamylphosphate. The reaction mixture was incubated 30 min at 37 °C.

Dihydroorotase Assay-- The DHOase activity was measured by the spectrophotometric method adapted by Minic et al. (9).

pH Dependence Assay-- The buffer system used to determine the pH dependence of the enzyme activity was composed of 51 mM diethanolamine, 51 mM N-ethylmorpholine, and 100 mM MES (22). The tri-buffer system covers the entire range of pH, without significant change of ionic strength. The pH dependence assay for UPase was carried out in 100 mM potassium phosphate.

Assays to Determine the Hydrolysis Products of CTP-- The hydrolysis products of CTP were determined by an adaptation of the protocol of Papas (23).

Each assay reaction mixture consisted of 50 mM Tris-HCl, pH 8.5, 5 mM MgCl2, 0.1 mM (0.2 mCi/mmol) [14C]CTP, and 10 mg of protein extract in a final volume of 1 ml. The reaction was carried out for 3 h at 37 °C and terminated by heating the reaction mixture to 90 °C for 60 s and immediate cooling in an ice bath. A 500-µl aliquot of reaction mixture was injected on a DEAE-Sephadex A-50 (Sigma) column (1.5 × 6 cm) to separate unhydrolyzed CTP from its hydrolyzed products. Products of hydrolysis were washed from the column with 5 mM Tris-HCl (pH 7.8), first alone and then with a discontinuous gradient of 0-0.4 M CH3COONH4. Individual fractions were counted for radioactivity, and the peaks for each fraction containing cytosine, CMP, CDP, and CTP were expressed as percent of total radioactivity.

Anion-exchange Chromatography-- The soluble protein extract of trophosome or vestimentum in a volume of 0.5-1.0 ml (20 mg) was loaded on a DEAE-Sepharose (Sigma) anion-exchange column (1.5 × 1.6 cm). Proteins were then eluted with 25 mM Tris-HCl in presence of 0.1% Triton X-100 (pH 7.8), first alone and then with a 0-0.5 M NaCl discontinuous gradient. One-ml fractions were collected and assayed for 5'-nucleotidase activity.

Protein Assay-- Total protein concentration was determined by the Lowry method (see Ref. 24), with bovine serum albumin dissolved in extraction buffer as the standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hydrolysis of CTP in the Different Parts of R. pachyptila-- In a first attempt to study the degradation of nucleotides in R. pachyptila, we tested the hydrolysis of CTP by the dialyzed cell-free extracts of various tissues of the worm. The results reported in Table I show the production of CDP, CMP, and cytidine in all tissues tested. The degradation to CMP can result from the presence of enzymes such as phosphatases and pyrophosphatases (25), whereas the hydrolysis of CMP into cytidine results from the action of a 5'-nucleotidase (12). These enzymes are present in all the tissues. The proportion of CMP and cytidine obtained with the trophosome extract suggests the presence of a high nucleotidase activity in this tissue. In contrast, by using the bacterial extract, only a very small degradation of CTP and no production of cytidine were observed, indicating that the bacterial symbiont would possess phosphatase and/or pyrophosphatase but no nucleotidase activity. On the basis of these results, we focused the analysis on 5'-nucleotidase and the two subsequent enzymes in the catabolism of pyrimidine nucleotides, UPase and uracilRase.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Determination of the CTP hydrolysis products in various body parts of R. pachyptila
Samples of 10 mg of dialyzed protein extracts of various body parts of R. pachyptila were incubated with 0.1 mM CTP at pH 8.5 for 3 h at 37 °C. The products of hydrolysis were separated by DEAE-Sephadex A-50 (see "Experimental Procedures" for technical details). The radioactivity of the cytosine, CMP, and CDP fractions obtained was measured, and each is expressed as percent of total radioactivity. ND, not detectable.

Distribution of Enzyme Activities of Pyrimidine Catabolism in the Different Tissues of R. pachyptila-- The results of analyses on the distribution of the activities of 5'-nucleotidase, UPase, and uracilRase are given in Table II. All three enzymatic activities were present in all tissues of the host. A very high UPase activity was found in the body wall. In contrast, this tissue exhibited very low 5'-nucleotidase activity. This 5'-nucleotidase activity was especially high in the trophosome. However, the isolated bacteria did not exhibit any activity for these enzymes of the catabolic pathway, in accordance with the result reported above for 5'-nucleotidase.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Activities of the specific enzymes that participate in the catabolism of pyrimidine nucleotides in the host tissues and isolated bacteria of R. pachyptila
See "Experimental Procedures" for the experimental details. All enzyme activities were measured at 37 °C. Assays for 5'-nucleotidase and uracilRase were performed in 50 mM Tris-HCl buffer, pH 8.0. UPase activity was determined in 100 mM potassium phosphate buffer, pH 7.9. Aliquots of 100 µl of isolated bacteria or protein extract (0.3-1.5 mg of protein) were used for the enzyme assays. The numbers in parentheses indicate the number of determinations made on different Riftia individuals. ND, not detectable.

Characterization of the Trophosomal Enzymes on the Basis of Their pH Dependence-- Although none of these enzymatic activities were detected in the bacterial extracts, we sought to obtain information on the origin of these three catabolic enzymes in the trophosome. The pH activity profiles of 5'-nucleotidase, UPase, and uracilRase determined in trophosome were compared with those of the enzymes present in another host tissue, the vestimentum. The results obtained are presented in Fig. 2. The profiles of UPase and uracilRase were identical in these two tissues. Taken together with the lack of these activities in the bacteria, these results indicate that these two enzymes belong to the host. In contrast, the pH profiles of 5'-nucleotidase in these two tissues were different, suggesting the presence of different enzymes. Consequently, the 5'-nucleotidase activity was further investigated.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2.   pH activity profiles of CMP-5'-nucleotidase, UPase, and uracilRase. Dialyzed crude extracts from the trophosome and the vestimentum (50-100 µl) were used to determine the pH dependence of the enzymatic activities. The tri-buffer system used for the pH activity profiles of 5'-nucleotidase and uracilRase contained 51 mM diethanolamine, 51 mM N-ethylmorpholine, and 100 mM MES. The UPase pH profile was determined in 100 mM potassium phosphate buffer. The activities were measured as described under "Experimental Procedures."

Analyses of 5'-Nucleotidase Activity in the Different Body Parts of R. pachyptila by Anion-exchange Chromatography-- The results presented above show important differences between the different tissues of Riftia in terms of 5'-nucleotidase activity (Table II). However, it is known that 5'-nucleotidase is strongly inhibited in crude protein extracts by physiological inhibitors (26-29). To determine whether the differences observed result from the presence of such inhibitors in some tissues, the 5'-nucleotidase activity was measured in the extracts of the various parts of Riftia after ion-exchange chromatography, a method that was shown previously to separate the enzyme and the inhibitors (26-29). The elution was performed in the presence of 0.1% of Triton X-100. Two peaks of 5'-nucleotidase activity were observed. Table III shows the activities corresponding to these two peaks that were detected in all tissues tested, except that peak II was absent in the branchial plume. It appears that under these conditions the highest 5'-nucleotidase activity was again found in the trophosome, particularly in peak II. However, 5'-nucleotidase activity was not observed in the absence of Triton X-100. This would indicate that the two detected 5'-nucleotidase peaks of activity belong to membrane proteins.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Proportion of the two peaks of 5'-nucleotidase activity in the different body parts of R. pachyptila
The protein extract of each body tissues of R. pachyptila (20 mg of protein) was chromatographed on a 1.5 × 1.6-cm DEAE-Sepharose column as reported in Fig. 3. The column was eluted discontinuously in 11 steps with 2.5-ml fractions of 25 mM Tris-HCl, pH 7.8, 0.1% Triton X-100 buffer containing increasing concentrations of NaCl from 0 to 0.5 M. Fractions of 1 ml were collected, and 100-µl aliquots were assayed for CMP-5'-nucleotidase activity, as described under "Experimental Procedures." ND, not detectable.

The presence of two peaks of activity is illustrated in Fig. 3 in the cases of trophosome and the vestimentum. In these experiments all the tissues exhibited the two peaks of 5'-nucleotidase at the same position, suggesting a common host origin for both enzymes.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3.   Elution profile of the anion-exchange chromatography of the trophosome and vestimentum extracts from R. pachyptila depicting the activity of 5'-nucleotidase. 0.5 ml of trophosome extract or 1.0 ml of vestimentum extract (20 mg protein each) was chromatographed on a 1 × 1.6-cm DEAE-Sepharose column. The column was eluted discontinuously in 11 steps with 2.5-ml fractions of 25 mM Tris-HCl, pH 7.8, 0.1% Triton X-100 buffer containing increasing concentrations of NaCl from 0 to 0.5 M. Fractions of 1 ml were collected, and 100-µl aliquots were assayed for CMP-5'-nucleotidase activity as described under "Experimental Procedures."

Since it was observed previously that the pH activity profiles of the 5'-nucleotidase present in the trophosome and in the vestimentum were different, the dependence on pH of the two peaks of 5'-nucleotidase activity was determined in the trophosome and in the vestimentum. Fig. 4 (A and B) shows that the corresponding peaks of the two types of tissues show the same pH profiles. These findings provide additional evidence that the two 5'-nucleotidase activities present in the trophosome belong to the host. The difference in the pH activity profiles of the trophosome and vestimentum crude extracts (Fig. 2) might result from the presence of different proportions of these two enzyme species in the extracts (Table III).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4.   pH dependence of 5'-nucleotidase activity. The 5'-nucleotidase activity was assayed in pooled fractions obtained after ion-exchange chromatography (see Fig. 3). A, peak I; B, peak II. The 5'-nucleotidase activity measurements were made using 100-µl aliquots of the chromatography fractions.

Catalytic Properties of 5'-Nucleotidase-- The activity of 5'-nucleotidase produces nucleosides, metabolites that can be used by both the pyrimidine and purine salvage metabolic pathways and the catabolic pathway. Such a key role could explain the high level of this enzyme found in the trophosome. Furthermore, it is well known that nucleoside monophosphates can be hydrolyzed not only by nucleotidases but also by nonspecific phosphatases (30-32). For this reasons we further investigated the catalytic activity of Riftia nucleotidase after separation by ion-exchange chromatography.

Substrate Specificity-- The two peaks of 5'-nucleotidase activity found in the trophosome extracts after ion-exchange chromatography were tested using a series of potential substrates.

The results obtained are shown in Table IV where it can be seen that these two activities do not have exactly the same specificity. Peak I had the highest relative activity using CMP as a substrate but also hydrolyzed other 5'-nucleotides. The best substrates for this enzymes are in order CMP > dCMP > OMP > UMP > GMP > IMP/dUMP > TMP > XMP > AMP. The enzyme of peak II had the highest relative activity toward AMP but also hydrolyzed other nucleotides. In this case the best substrates are in the order AMP > GMP > dUMP > CMP > UMP > IMP > XMP > dCMP > TMP > OMP. Clearly these two enzymes show different specificities and the activity of peak II shows some preference for purine nucleotide monophosphates.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Substrate specificity of the two peaks of 5'-nucleotidase activity obtained after DEAE-Sepharose chromatography
Aliquots of 200 µl were assayed for 5'-nucleotidase activity in the presence of 1 mM of each substrate. The reaction was stopped by the addition of 200 µl of 10% trichloroacetic acid after 30 min, and the liberated phosphate was determined colorimetrically. The control was an incubation mixture at time 0. Results are expressed as a percentage of the specific activity obtained with CMP as substrate in 50 mM Tris-HCl buffer, pH 8.0.

It is important to note that p-nitrophenyl phosphate, a substrate for phosphatase, was not hydrolyzed either by peak I or by peak II ensuring that such a phosphatase does not interfere with our determinations.

Substrate Saturation Curves-- The substrate saturation curves for UMP and CMP of the enzymes present in peaks I and II were determined. Fig. 5 shows the results obtained and the kinetic parameters resulting from their fit to the Michaelis-Menten equation. For UMP and CMP, respectively, in the case of peak I, Km values of 67 ± 9 and 219 ± 13 µM were obtained (Fig. 5A). For peak II, Km values of 45 ± 4 and 108 ± 8 µM were obtained (Fig. 5B). These differences confirm that these two peaks of activity correspond to different enzymes.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 5.   UMP and CTP saturation curves of 5'-nucleotidase. Aliquots of 50 µl from peak I (A) and peak II (B) pooled fractions were used for the assay of 5'-nucleotidase activity in a total volume of 200 µl. Reaction mixtures were incubated at 37 °C for 30 min and contained increasing concentrations of UMP or CMP from 7 to 1050 µM. The insets present the double-reciprocal plots used for the determination of Km.

Inhibition-- To characterize further these two 5'-nucleotidase activities, they were examined in the presence of various compounds known to be inhibitors of 5'-nucleotidase (33-38). The inhibitory effects of these inhibitors at a 2 mM concentration on the dephosphorylation of CMP are shown in Fig. 6. It appears that 1 mM EDTA provokes the complete inhibition of the two enzymes; this result indicates that these 5'-nucleotidases use Mg2+ as a cofactor, as do the homologous enzymes described previously (39, 40). The activity was restored by addition of 5 mM Mg2+ (not shown). At a concentration of 2 mM the other compounds tested had increasing inhibitory effects in the order ATP > ribulose 5'-phosphate/cAMP > inosine. The two enzymes were similarly inhibited by these various compounds. The two nucleotidase activities are insensitive to NaF, a strong inhibitor of phosphatase (41), confirming the lack of phosphatase activity in these preparations.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6.   Influence of various effectors on the activity of 5'-nucleotidases. The nucleotidase activity was measured using the pooled fractions of peak I and peak II coming from the DEAE-Sepharose chromatography of the trophosome (see Fig. 3) under standard conditions in the presence of ATP, cAMP, inosine, ribulose 5'-phosphate, EDTA, and NaF.

In addition, kinetic analyses were performed to determine the type of inhibition provoked by each of these compounds. These experiments were carried out using 1, 2, and 4 mM fixed concentration of inhibitors. The results obtained are shown in Fig. 7. For both peaks of activity, the inhibition by ATP and ribose 5'-phosphate were competitive, whereas the inhibition by inosine was uncompetitive. Cyclic AMP was a noncompetitive inhibitor for peak I and partial noncompetitive inhibitor for peak II.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 7.   Lineweaver-Burk plots of 5'-nucleotidase activities in the presence of various inhibitors. CMP saturation curves of peak I and peak II coming from DEAE-Sepharose chromatography (see Fig. 3) were used for nucleotidase activity measurements in the presence of the different inhibitors, using 100-µl aliquots. As indicated, two concentrations of inhibitor were used, i.e. 2 and 4 mM in the case of ATP, ribulose 5'-phosphate, and inosine and 1 and 2 mM in the case of cAMP. Enzyme activity was determined as described under the "Experimental Procedures." Rib-5'-P, ribulose 5'-phosphate.

These catalytic properties and inhibitor effects are characteristic of 5'-nucleotidase and are similar to those found in the case of other animal sources (42-44).

Heterogeneity of Localization of Pyrimidine Metabolism Enzymes in the Trophosome-- Analysis of the trophosome by electron microscopy has suggested the presence of the symbiont at various states of development as follows: dividing bacteria predominantly present in the central part of the trophosome lobules (clusters of bacteriocytes) and lysed bacteria present at the periphery (45). We hypothesized that such a specific tissue organization could exist across the entire trophosome, accompanied by differential distribution of the enzymes of catabolism and biosynthesis of pyrimidine nucleotides.

To test this hypothesis, the distribution of enzyme activities in different parts of the trophosome was investigated from the center of the trophosome to its periphery. The two catabolic enzymes, 5'-nucleotidase and UPase and two enzymes of the anabolic pyrimidine biosynthetic pathway, ATCase and DHOase, were tested in three zones of the trophosome tissue (center, middle, and periphery as indicated in Fig. 1). The results obtained are given in Fig. 8. The distribution of peaks I and II activities of 5'-nucleotidase and UPase shows that maximal enzyme activity is found in the periphery of the trophosome. In contrast, ATCase and DHOase activities were the highest in the central zone. Thus, there seems to be opposite gradients of the activities of the catabolic and anabolic enzymes of the pyrimidine nucleotides pathway in the trophosome.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 8.   Variations of anabolic and catabolic enzymatic activities across the trophosome. The activities of 5'-nucleotidase, UPase, ATCase, and DHOase were determined in the center, middle, and periphery of cross-sections of the trophosome. These activities were measured under the conditions described under "Experimental Procedures" at 37 °C. Assays for 5'-nucleotidase and ATCase were performed in 50 mM Tris-HCl buffer, pH 8.0. The DHOase activity was determined in 100 mM potassium buffer, pH 6.5. The 5'-nucleotidase activity was determined after DEAE-Sepharose column chromatography as described in Fig. 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have investigated the distribution and properties of enzymes involved in the catabolism of pyrimidine nucleotides in R. pachyptila. The results obtained, together with data reported previously (9) on the de novo and salvage pathways, allow us to propose a general scheme for the organization of the metabolism of pyrimidine nucleotides in R. pachyptila and its endosymbiont (Fig. 9). This scheme assembles the results described below.


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 9.   Integrated scheme of the metabolic pathways of pyrimidine nucleotides in R. pachyptila and its bacterial endosymbiont. The model is based on the distribution and properties of enzymes of the pyrimidine nucleotide catabolism presented in this work and on the results reported previously (9) for de novo and salvage pathways for biosynthesis of the pyrimidine nucleotides. The model describes the exchanges between the endosymbiont, the trophosomal host cells, and cells of other host tissues of R. pachyptila. Question marks indicate steps that have not been completely elucidated. Thin arrows refer to metabolic pathways. Thick arrows refer to transport of metabolites in compartments, tissues of body parts.

The symbiotic bacteria possesses enzymatic equipment for the biosynthesis of pyrimidine nucleotides through the de novo pathway but lacks the enzymes of the salvage (9) and catabolic pathways as demonstrated here. In contrast, the host cells (including the bacteriocytes) in R. pachyptila possess the enzymes catalyzing the final steps of the de novo pathway as well as the enzymatic equipment for the salvage pathways leading to the synthesis of pyrimidines from nucleic acid degradation products. Since the host cells do not have the first three enzymes of the de novo pathway (carbamylphosphate synthetase, ATCase, and DHOase), the necessary metabolic precursors, orotate and/or dihydroorotate, must be provided by the bacteria. The first reaction of this de novo pathway (carbamyl-phosphate synthetase) relies on inorganic carbon and nitrogen provided by the external medium. Thus, the de novo pathway to pyrimidine nucleotides in R. pachyptila is absolutely dependent on the symbiotic bacteria. For this reaction to occur, CO2, NH3, and nitrate are provided by the external environment. Nitrate is reduced by assimilatory enzymes present only in the bacteria (9, 46-48). The resulting NH3 is used for the synthesis of glutamine from glutamate; glutamine is the substrate of the carbamyl-phosphate synthetase specific to the pyrimidine pathway and present only in the bacteria (9, 10).

The results reported here show that R. pachyptila possesses the activities of three enzymes participating in the catabolism of pyrimidine nucleotides, 5'-nucleotidase, UPase, and uracilRase, in all its tissues. Notably, these enzymes do not exist in the bacterial endosymbiont. Catabolism of pyrimidine nucleotides leads to the production of CO2, NH3, malonyl-CoA, and succinyl-CoA; subsequently malonyl-CoA can be used for the biosynthesis of fatty acids, whereas succinyl-CoA enters into the cycle of citric acid (15, 49, 50). In this manner the degradation of pyrimidine nucleotides can represent an alternative nutritional source of nitrogen and carbon, besides the external environment of the worm, and thus can feed other biosynthetic pathways. Indeed, previous observations (51, 52) indicate that the supply of inorganic carbon and nitrogen must limit the growth of Riftia. In the case of carbon, the CO2 concentration around the Riftia tube is about 5 mM (51), and it was emphasized that the worm metabolism implies a high demand for inorganic carbon whose concentration must be limiting inside the symbiont (51, 52). In the case of nitrogen the concentrations of nitrate and ammonia around the tubeworm are 15-40 and 0.1 to 0.3 µM, respectively, concentrations which are also considered as limiting (53). Under these growth conditions an alternative source of inorganic carbon and nitrogen would contribute to the development of the worm. The effectiveness of the catabolism of nucleosides in providing carbon and nitrogen was well documented in a series of microorganisms (49, 54) and in mammals (55). In the case of Tetrahymena pyroformis, it was shown that 50% of the degradation products of labeled thymidine was recovered in macromolecules other than nucleic acids (56).

As far as 5'-nucleosidase is concerned, the highest activity was found in the trophosome, suggesting that the production of nucleosides is mainly occurring in trophosomic cells. Related to this, it has been recently reported by De Cian et al. (57) that a particularly high concentration of purine nucleoside (guanosine and inosine) is present in the trophosome tissue. Our results have shown that after chromatographic separation, two peaks of 5'-nucleotidase activity are found that catalyze the hydrolysis of both GMP and IMP into guanosine and inosine, respectively. Contrary to nucleotides, which cannot cross the cell membranes, nucleosides (uridine, cytidine, uracil, guanosine, inosine, etc.) can traverse membranes (11) and could thereby be delivered to other tissues of the worm. In this manner, the nucleosides generated in the trophosome from the degradation of nucleic acids (including those of the bacteria) could be transported to the other parts of the host and be used in both the salvage and the catabolic pathways.

Ultrastructural cellular studies have shown the occurrence of symbiont lysis in the trophosome (45, 58, 59), and it was proposed that such lysis might represent a nutritional source for R. pachyptila. Our enzymatic analysis shows the existence of a gradual increase of the catabolic enzyme activities, 5'-nucleotidase and UPase, from the center toward the peripheral region of the trophosome. In contrast, the activity of the bacterial enzymes that participate in the de novo biosynthesis of pyrimidine nucleotides, ATCase and DHOase, decreases from the center of the trophosome to its periphery. This observation is consistent with the hypothesis that an important production of nucleosides from nucleotides would derive from lysed bacteria predominantly in the periphery of the trophosome. On the other hand, the central zone of the trophosome would contain a high population of actively dividing bacteria. It is well known that the enzymatic activities of the de novo pyrimidine pathway are especially high in rapidly dividing cell populations (60). These observations emphasize the idea that the trophosome is not a homogeneous tissue but one that exhibits a structural and physiological organization. The specific tissue distribution of hydrolytic enzymes, such as 5'-nucleotidase and UPase, across the trophosome may be part of an organized hydrolytic system compensating the fact that R. pachyptila does not have a differentiated digestive system.

In conclusion, the two symbiotic partners in R. pachyptila have developed a particular metabolic organization and a nutritional strategy involving numerous interactions and metabolic exchanges as shown here in the particular case of pyrimidine metabolism. This complex organization is the basis of the adaptation of R. pachyptila to the extreme hydrothermal vent environment and the absence of readily available sources of organic carbon through photosynthesis.

    ACKNOWLEDGEMENTS

We thank the skillful and enthusiastic crews of the oceanographic ship ATLANTE and of the submarine NAUTILE of Institut Française de Recherche et l'Exploitation des Mers.

    FOOTNOTES

* This work was supported by the CNRS, l'Université Pierre et Marie Curie, and a grant from the program "DORSALES" of the Institut National des Sciences de l'Univers.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, UMR 7631, CNRS, Université Pierre et Marie Curie, 96 Blvd. Raspail, F-75006 Paris, France. Tel.: 33 1 53 63 40 70; Fax: 33 1 42 22 13 98; E-mail: gherve@ccr.jussieu.fr.

Published, JBC Papers in Press, October 8, 2001, DOI 10.1074/jbc.M108035200

    ABBREVIATIONS

The abbreviations used are: ATCase, aspartate transcarbamylase; DHOase, dihydroorotase; MES, 2(N-morpholino)ethanesulfonic acid; uracilRase, uracil reductase; UPase, uridine phosphorylase; NPP, p-nitrophenyl phosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Gaill, F. (1993) FASEB J. 7, 558-565[Abstract]
2. Jones, M. L. (1981) Proc. Biol. Soc. Wash. 93, 1295-1313
3. Jones, M. L. (1981) Science 213, 333-336[Abstract/Free Full Text]
4. Cavanaugh, C. M., Gardiner, S. L., Jones, M. L., Jannasch, H. W., and Waterbury, J. B. (1981) Science 213, 340-342[Abstract/Free Full Text]
5. Felbeck, H. (1981) Science 213, 336-338[Abstract/Free Full Text]
6. Nelson, D. C., and Fisher, C. R. (1995) in The Microbiology of Deep-sea Hydrothermal Vents (Karl, D. M., ed) , pp. 125-167, CRC Press, Inc., Boca Raton, FL
7. Powell, M. A., and Somero, G. N. (1986) Biol. Bull. 171, 274-290[Abstract/Free Full Text]
8. Zal, F., Lallier, F. H., Wall, J. S., Vinogradov, S. N., and Toulmond, A. (1996) J. Biol. Chem. 271, 8869-8874[Abstract/Free Full Text]
9. Minic, Z., Simon, V., Penverne, B., Gaill, F., and Hervé, G. (2001) J. Biol. Chem. 276, 23777-23784[Abstract/Free Full Text]
10. Simon, V., Purcarea, C., Sun, K., Joseph, J., Frebourg, G., Lechaire, J. P., Gail, F., and Hervé, G. (2000) Mar. Biol. (Berlin) 136, 115-127[CrossRef]
11. Berlin, R. D., and Olivier, J. M. (1975) Int. Rev. Cytol. 42, 287-336[Medline] [Order article via Infotrieve]
12. Zimmermann, H. (1992) Biochem. J. 285, 345-365
13. Niedzwicki, J. G., el Kouni, M. H., Chu, S. H., and Cha, S. (1983) Biochem. Pharmacol. 32, 399-415[CrossRef][Medline] [Order article via Infotrieve]
14. Podschun, B., Wahler, G., and Schnackerz, K. D. (1989) Eur. J. Biochem. 185, 219-224[Medline] [Order article via Infotrieve]
15. Borel, J. P., Maquart, F. X., Le, Peuch, C., Randoux, A., Gillery, P., Bellon, G., and Monboisse, J. C. (1997) Biochimie Dynamique , pp. 773-785, De Boeck Université, Paris
16. Distel, D. L., and Felbeck, H. (1987) Mar. Biol. (Berlin) 96, 97-106
17. West, J. T. C., and Beutler, E. (1977) J. Lab. Clin. Med. 90, 563-568[Medline] [Order article via Infotrieve]
18. Ellims, P. H., Bailey, L., and Van der Weyden, B. (1978) Clin. Chim. Acta 88, 99-103[CrossRef][Medline] [Order article via Infotrieve]
19. Niedzwiecka, J., and Jaroszewicz, L. (1996) Biochem. Biophys. Acta 221, 471-476
20. Scocca, J. J. (1978) Methods Enzymol. 51, 432-437[Medline] [Order article via Infotrieve]
21. Schmitt, U., Jahnke, K., Rosenbaum, K., Cook, P. F., and Schnackerz, K. D. (1996) Arch. Biochem. Biophys. 332, 175-182[CrossRef][Medline] [Order article via Infotrieve]
22. Léger, D., and Hervé, G. (1988) Biochemistry 27, 4293-4298[CrossRef]
23. Papas, P. W. (1983) Mol. Biochem. Parasitol. 8, 1-16[CrossRef][Medline] [Order article via Infotrieve]
24. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
25. Zimmermann, H. (2000) Naunyn-Schmiedeberg's Arch. Pharmacol. 362, 299-309[CrossRef][Medline] [Order article via Infotrieve]
26. Neu, H. C. (1967) J. Biol. Chem. 242, 3905-3911[Abstract/Free Full Text]
27. Uchino, K., Ogawara, H., Akiyama, T., Fukuchi, A., Shibata, S., Takahashi, K., and Narui, T. (1985) J. Antibiot. (Tokyo) 38, 157-160[Medline] [Order article via Infotrieve]
28. Rajput, A., Chakravarthy, B. R., and D'Souza, C. J. (1992) Indian J. Biochem. Biophys. 29, 204-208[Medline] [Order article via Infotrieve]
29. Torrance, J. D., Whittaker, D., and Beutler, E. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3701-3704[Abstract/Free Full Text]
30. Jensen, M. H., Iversen, A., and Hägerstrand, I. (1980) Clin. Chim. Acta 104, 221-226[CrossRef][Medline] [Order article via Infotrieve]
31. Beutler, E., and West, C. (1982) Biochem. Med. 27, 334-341[CrossRef][Medline] [Order article via Infotrieve]
32. Johnson, S., and Pitcher, D. (2000) FEMS Microbiol. Lett. 192, 59-65[Medline] [Order article via Infotrieve]
33. Paglia, D. E., and Valentine, W. N. (1975) J. Biol. Chem. 250, 7973-7979[Abstract/Free Full Text]
34. Polya, G. M. (1975) Biochim. Biophys. Acta 384, 443-457[Medline] [Order article via Infotrieve]
35. Burger, R. M., and Lowenstein, J. M. (1975) Biochemistry 14, 2362-2366[CrossRef][Medline] [Order article via Infotrieve]
36. Naito, Y., and Tsushima, A. K. (1976) Biochim. Biophys. Acta 438, 159-168[Medline] [Order article via Infotrieve]
37. Camici, M., Fini, C., and Ipata, P. L. (1985) Biochim. Biophys. Acta 840, 6-12[Medline] [Order article via Infotrieve]
38. Madrid-Mariana, V., and Fox, I. H. (1986) J. Biol. Chem. 261, 444-452[Abstract/Free Full Text]
39. Mallol, J., and Bozal, J. (1983) J. Neurochem. 40, 1205-1211[CrossRef][Medline] [Order article via Infotrieve]
40. Grondal, E. J. M., and Zimmermann, H. (1987) Biochem. J. 245, 805-810[Medline] [Order article via Infotrieve]
41. Campbell, T. A., Zlotnick, G. W., Neubert, T. A., Sacci, J. B., Jr., and Gottlieb, M. (1991) Mol. Biochem. Parasitol. 47, 109-117[CrossRef][Medline] [Order article via Infotrieve]
42. Fritzson, P. (1969) Biochim. Biophys. Acta 178, 534-541[Medline] [Order article via Infotrieve]
43. Bosmann, H. B., and Pike, G. Z. (1971) Biochim. Biophys. Acta 227, 402-412[Medline] [Order article via Infotrieve]
44. El Kouni, M. H., and Cha, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1037-1041[Abstract/Free Full Text]
45. Bright, M., Keckeis, H., and Fisher, C. R. (2000) Mar. Biol. (Berlin) 136, 621-632[CrossRef]
46. Hentschel, U., and Felbeck, H. (1993) Nature 366, 338-340[CrossRef]
47. Lee, R. W., Robinson, J. J., and Cavanaugh, C. M. (1999) J. Exp. Biol. 202, 289-300[Abstract]
48. Girguis, P. R., Lee, R. W., Desaulniers, N., Childress, J. J., Pospesel, M., Felbeck, H., and Zal, F. (2000) Appl. Environ. Microbiol. 66, 2783-2790[Abstract/Free Full Text]
49. Milstein, O. A., and Bekker, M. L. (1976) J. Bacteriol. 127, 1-6[Abstract/Free Full Text]
50. Masood, R., and Venkitasubramanian, T. A. (1987) Ann. Inst. Pasteur Microbiol. 138, 501-507[CrossRef][Medline] [Order article via Infotrieve]
51. Childress, J. J., Lee, R. W., Sanders, N. K., Felbeck, H., Oros, D. R., Toulmond, A., Desbruyeres, D., Kennicutt, M. C., II, and Brooks, J. (1993) Nature 362, 147-149[CrossRef]
52. Scott, K. M., Fisher, C. R., Vodenichar, J. S., Nix, E. R., and Minnich, E. (1994) Physiol. Zool. 67, 617-638
53. Lee, R. W., and Childress, J. J. (1996) Biol. Bull. 190, 373-384[Abstract]
54. West, T. P. (1997) Arch. Microbiol. 168, 237-239[CrossRef]
55. Sonada, T., and Tatibana, M. (1978) Biochim. Biophys. Acta 521, 55-66[Medline] [Order article via Infotrieve]
56. Niemann, M. A., and Berech, J., Jr. (1981) Biochim. Biophys. Acta 652, 347-353[Medline] [Order article via Infotrieve]
57. De Cian, M.-C., Regnault, M., and Lallier, F. H. (2000) J. Exp. Biol. 203, 2907-2920[Abstract]
58. Gardiner, S. L., and Jones, M. L. (1993) in Microscopic Anatomy of Invertebrates (Harrison, F. W. , and Rice, M. E., eds) , pp. 371-460, Wiley-Liss, Inc., New York
59. Bosch, C., and Grassé, P. P. (1984) C. R. Acad. Sci. III 299, 371-376 Paris
60. Hervé, G., and Xi, X. G. (1991) in Mécanismes des Pneumopathies Professionnelles (Sebastien, P., ed) , pp. 195-206, INSERM, Paris

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
S. S. Orchard and H. Goodrich-Blair
Pyrimidine Nucleoside Salvage Confers an Advantage to Xenorhabdus nematophila in Its Host Interactions
Appl. Envir. Microbiol., October 1, 2005; 71(10): 6254 - 6259.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Minic and G. Herve
Arginine Metabolism in the Deep Sea Tube Worm Riftia pachyptila and Its Bacterial Endosymbiont
J. Biol. Chem., October 17, 2003; 278(42): 40527 - 40533.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/1/127    most recent
M108035200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Minic, Z.
Right arrow Articles by Hervé, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Minic, Z.
Right arrow Articles by Hervé, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement