Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M501805200 on February 28, 2005

J. Biol. Chem., Vol. 280, Issue 17, 16748-16753, April 29, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/17/16748    most recent
M501805200v1
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 Roessner, C. A.
Right arrow Articles by Scott, A. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Roessner, C. A.
Right arrow Articles by Scott, A. I.
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?

Genetically Engineered Production of 1-Desmethylcobyrinic Acid, 1-Desmethylcobyrinic Acid a,c-Diamide, and Cobyrinic Acid a,c-Diamide in Escherichia coli Implies a Role for CbiD in C-1 Methylation in the Anaerobic Pathway to Cobalamin*{boxs}

Charles A. Roessner{ddagger}, Howard J. Williams, and A. Ian Scott

From the Center for Biological NMR, Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255

Received for publication, February 17, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiA, -C, -D, -E, -T, -F, -G, -H, -J, -K, -L, and -P genes from Salmonella enterica serovar typhimurium in Escherichia coli resulted in the production of cobyrinic acid a,c-diamide. A cbiD deletion mutant of this strain produced 1-desmethylcobyrinic acid a,c-diamide, indicating that CbiD is involved in C-1 methylation in the anaerobic pathway to cobalamin. Strains that did not have the cbiP gene also produced 1-desmethylcobyrinic acid a,c-diamide, and strains that had neither cbiP nor cbiA synthesized 1-desmethylcobyrinic acid even in the presence of cbiD, suggesting that CbiA and CbiP are necessary for CbiD activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
There are two recognized pathways to cobalamin (1), one that requires molecular oxygen (aerobic pathway) and one that does not (anaerobic pathway). The intermediates of the aerobic route, from aminolevulinic acid (ALA)1 to adenosylcobalamin in Pseudomonas denitrificans, have all been biosynthesized and their structures determined (1, 2). In addition, the role of cobalt in early intermediates of the anaerobic pathway and its importance in the mechanism of ring contraction in the absence of oxygen were established (3) in a cell-free system. In this system, precorrin 3 was produced from uroporphyrinogen III and then converted to cobalt-precorrin 4 (Fig. 1) by non-enzymatic cobalt chelation and C-17 methylation (with the concomitant ring contraction) by CbiH of Salmonella enterica serovar typhimurium LT2. However, the in vitro synthesis of those intermediates of the anaerobic pathway, which have been proposed to lie between cobalt-precorrin 4 and cobyrinic acid (Fig. 1), has proved difficult, and most of their structures remain unknown. Furthermore, the enzymes CbiD and CbiG, which are found in almost all organisms that utilize the anaerobic pathway and were shown previously to be essential for adenosyl-cobyric acid biosynthesis (4, 5), have not been functionally characterized. Although CbiG shows some similarity to the P. denitrificans CobE protein (function not known), CbiD has no known counterpart in the aerobic pathway. Because it has a potential S-adenosyl-L-methionine binding site (6), CbiD was suggested previously to be the C-1 methyltransferase (7), but no experimental data have yet been provided to support this proposal. In the aerobic pathway, the C-1 methyltransferase is encoded by the cobF gene (8), but an analog of cobF has been reported in only one of the organisms that utilize the anaerobic pathway to cobalamin. The recent sequence of the Propionibacterium acnes genome (9) revealed the presence of proteins similar to CobF and CbiG but none that showed homology to CbiD, suggesting that CobF takes the place of CbiD in this organism.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 1.
The proposed anaerobic pathway from uroporphyrinogen III to adenosyl-cobyric acid. The structures of the intermediates shown in brackets have not been confirmed. SAM, S-adenosyl-L-methionine.

 
To provide an experimental system for analysis of the function of CbiD, we engineered new strains of Escherichia coli that bear cobalamin biosynthetic genes from the anaerobic pathway of S. enterica. One of these strains produced cobyrinic acid a,c-diamide, whereas other strains produced related compounds. These compounds, however, lacked the C-1 methyl group, a phenomenon not observed previously in cobalamin biosynthesis but one that provides the first evidence that CbiD is involved in C-1 methylation during the biosynthesis of this cofactor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—ALA was purchased from Sigma. [13C]ALA was prepared as described previously (10). L-[methyl-13C]Methionine was purchased from Cambridge Isotope Laboratories. Restriction enzymes and T4 DNA ligase were from New England Biolabs. PCR primers and DNA sequencing services were provided by the Gene Technologies Laboratory (Biology Department, Texas A&M University). Plasmids and strains were constructed as outlined in Table I using standard molecular biology techniques (11).


View this table:
[in this window]
[in a new window]
  		TABLE I
Strains and plasmids used in this study

 
Cell Growth, Isolation, and Characterization of Products—E. coli strains were grown with aeration at 37 °C to an A600 = 1 in LB medium containing 0.2% glucose and the appropriate antibiotics (50 mg/liter ampicillin, 30 mg/liter chloramphenicol, 50 mg/liter kanamycin), and the cells were collected by centrifugation. The combined cell pellets from 4 liters of LB were resuspended in 1 liter of minimal medium, which consisted of M9 salts containing the same antibiotics plus the following additives: 0.2% glycerol, 0.2% L(+)-arabinose, 0.01% yeast extract, 2 mM MgCl2, 0.1 mM CaCl2, 20 mg of CoCl2 {diamondsuit}6H2O, 20 mg of ALA, and (for strains containing pZS*24-based plasmids) 0.2 mM isopropyl-{beta}-D-thiogalactopyranoside. Products for 13C NMR analysis were prepared by adding either 10–20 mg of [13CxALA instead of unlabeled ALA to label the ring or 50 mg of [13C]methionine to label the S-adenosyl-L-methionine-derived methyl groups. The cells in minimal medium were incubated with aeration at 37 °C for 24 h and without aeration at 25 °C for 24 h, collected by centrifugation, resuspended in 100 ml of 50 mM Tris-HCl, and lysed by sonication. The porphyrinoids found in both the culture medium and in the cell lysate were isolated by binding them to DEAE-Sephadex followed by esterification in methanol:sulfuric acid (95:5) and separation by thin layer chromatography on silica plates (3). Solid KCN was added to the product and to the solvent (dichloromethane:methanol, 95:5) prior to TLC. The isolation procedure resulted in purification of the products in the cyano-methylester form, which were extracted from the silica gel with the TLC solvent and quantitated using {epsilon}368 = 3.04 x 104 M–1 cm–1.

Spectral Analyses—UV-visible spectra were obtained with an Ocean Optics USB2000 miniature fiber optic spectrometer. 13C NMR spectra were acquired on a Bruker ARX 500 spectrometer at 125 MHz using a 5-mm C/H probe with a 1-s relaxation delay, a 30° pulse, accumulation of 40,000 scans, and Fourier transformation with 1-Hz line broadening. The samples were dissolved in KCN-saturated C6D6, and the benzene central line (128 ppm) was used for calibration. Mass spectral analyses were provided by the Laboratory for Biological Mass Spectrometry (Chemistry Department, Texas A&M University).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Production of 1-Desmethylcobyrinic Acid in E. coli—The first product analyzed was isolated from strain CR681, which was constructed to contain all of the genes believed to be necessary to convert uroporphyrinogen III to cobyrinic acid (Fig. 1, the enzymes that convert ALA to uroporphyrinogen III are provided by the host). The esterified product isolated from CR681 was similar to cobyrinic acid heptamethylester (cobester) based on Rf (see TLCs in the supplemental material), UV-visible spectroscopy (Fig. 2), and 13C NMR spectrometry of the product derived from 13C-enriched ALA (Table II and the NMR spectra in the supplemental material). However, the chemical shifts of some of the ring carbons differed significantly from those expected for cobester, especially that of C-19 (a 3.4-ppm difference). In addition, the mass of the product was 14 daltons less than that expected for cobester, which could be accounted for by loss of a methyl group. When the cells were grown on [13C]methionine to label the methyl groups, the chemical shifts of the six labeled methyl groups did not correspond to the seven S-adenosyl-L-methionine-derived methyl groups of cobester reported for cobester; especially conspicuous was the lack of the downfield signal at around 22.4 ppm corresponding to the C-1 methyl group. Further NMR analyses (see DEPT90, two-dimensional heteronuclear single quantum correlation proton-carbon correlation, and heteronuclear multiple band correlation spectra in the supplemental material) showed beyond a doubt that C-1 of the CR681 product was protonated rather than methylated, proving that the isolated product was, indeed, 1-desmethylcobester (Fig. 3).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2.
UV-visible spectra of authentic cobester (1), the product isolated from strain CR681 containing all of the genes for cobyrinic acid biosynthesis (2), and the product isolated from CR701 (cbiD–) (3).

 


View this table:
[in this window]
[in a new window]
  		TABLE II
NMR, mass, and Rf characterization of the esterified products isolated from engineered strains

 



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3.
The structure of 1-desmethylcobyrinic acid.

 
CbiG Is Essential for 1-Demethyl Cobyrinic Acid Synthesis—A cbiG– strain (CR694) was constructed by removal of a 437-base pair BstBI fragment from pCAR679, which resulted in the deletion of residues 156–291 within the second half of the 341-amino acid CbiG protein, including the region similar to CobE, and created a fusion protein consisting of the remainder of CbiG and all of CbiH. No corrinoid products could be isolated from the cbiG deletion mutant (see TLC in the supplemental material). Evidence that the truncated CbiG/CbiH fusion protein still had CbiH activity and that no polar effects were created by deletion of the BstBI fragment was provided by transforming a third plasmid, pCAR698, which bears a complete cbiG gene, into CR694. Cobyrinic acid synthesis in this strain, CR699, was rescued by complementation of the mutant cbiG gene by pGEC4 (see TLC in the supplemental material).

CbiD Is Not Necessary for 1-Demethyl Cobyrinic Acid Biosynthesis—A cbiD– strain (CR701) was constructed by removing a 1060-base pair SnaBI-SalI fragment from the plasmid bearing the cbiC-J genes, which resulted in the loss of 93% of the 379-amino acid CbiD protein. This strain, otherwise identical to CR681, also produced 1-desmethylcobyrinic acid based on UV-visible, NMR, and mass analyses (Fig. 2, Table II), suggesting that CbiD is necessary for C-1 methylation.

Production of 1-Desmethylcobyrinic Acid a,c-Diamide— Based on the homology of CbiA with CobB of the aerobic pathway, CbiA is the enzyme that amidates the a and c side chains of cobyrinic acid to afford cob(II)yrinic acid a,c-diamide, which is then reduced to cob(I)yrinic a,c-diamide and adenosylated to yield adenosyl-cobyrinic acid a,c-diamide (Fig. 1). Thus, the cbiA gene was inserted into pZS*24 and the plasmid transformed into strains CR681 and CR701 to afford CR716 and CR718. An identical product was isolated from both strains with Rf and mass values (Table II and TLC in supplemental material) lower than those of the CR681 and CR701 product and consistent with the bisamidation of 1-desmethylcobyrinic acid. Although the amidation sites have not been confirmed, the assumption is made that the a and c side chains of the product are amidated, thus affording 1-desmethylcobyrinic acid a,c-diamide.

Production of Cobyrinic Acid a,c-Diamide—Based on its homology with CobQ of the aerobic pathway, CbiP is proposed to be the enzyme that amidates the b, d, e, and g side chains of adenosyl-cobyrinic acid a,c-diamide to afford adenosyl-cobyric acid (Fig. 1). In addition, previous reports (4, 5) have indicated that E. coli, when grown anaerobically, has an endogenous system that can convert cobyrinic acid a,c-diamide to adenosylcobyric acid when CbiP is present. To test whether our engineered E. coli strain could further process 1-demethyl cobyrinic acid a,c-diamide, the cbiP gene was introduced into the plasmid bearing cbiA and transformed into CR681 and CR701 to afford strains CR727 and CR728. Two products that bound to DEAE-Sephadex were isolated from CR727 (see TLC in the supplemental material). The mass and 13C NMR spectrum (derived from 13C-4 ALA) of the product in the lower band extracted from the TLC plate corresponded to 1-desmethylcobester a,c-diamide, and, surprisingly, the mass and 13C NMR spectra (derived from 13C-4 ALA and [13Cxmethionine, Table II) of the product in the upper band corresponded to cobester a,c-diamide. The presence of the C-1 methyl group in this product has been confirmed by two-dimensional NMR analyses (see NMR spectra in the supplemental material). On the other hand, the mass and the methyl group NMR spectrum of the product isolated from CR728 (identical to CR727 except cbiD–) correspond to that of 1-desmethylcobester a,c-diamide, and a strain (CR735) in which only the cbiP gene was transformed into CR681 produced 1-demethyl cobyrinic acid. The product isolated from each strain described in this paper is summarized in Table III.


View this table:
[in this window]
[in a new window]
  		TABLE III
Products isolated from genetically engineered strains of E. coli

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have described several genetically engineered strains of E. coli that synthesize corrinoid products. One strain, CR727, synthesizes cobyrinic acid a,c-diamide, whereas another strain, CR728, which differs only in the lack of the cbiD gene, synthesizes 1-desmethylcobyrinic acid a,c-diamide. This result alone provides solid evidence that CbiD is either itself the C-1 methyltransferase or performs some role prerequisite to C-1 methylation. A paradox exists, however, in that the products isolated from other strains that do have the cbiD gene are also unmethylated at the C-1 position. These strains have either no additional genes (CR681) or one of the two amidases cbiA or cbiP (CR716 and CR735, respectively). It is possible that in these strains, cbiD is the only gene not expressed from the large plasmid or that CbiD, when overexpressed, is not folded correctly or is insoluble. Varying the growth conditions (lower temperatures, no aeration) had no effect on the CR681 product (not shown). To express cbiD separately from the other genes in the large plasmid (and because enzymes from different sources are sometimes more soluble), the cbiD genes from not only S. enterica but also Pseudomonas aeruginosa and Methanocaldococcus jannaschii were ligated into pZS*24, and the resultant plasmids were transformed into strain CR681. The three resultant strains all still produced 1-desmethylcobyrinic acid (not shown). Because neither CbiA nor CbiP alone is sufficient for CbiD activity (strains CR716 and CR735), it appears that CbiD, even though it may be produced, is not active unless both CbiA and CbiP are present. It could be that these two proteins act as a chaperone for the proper folding of CbiD or that the three enzymes (perhaps along with other enzymes of the pathway) form a complex, and CbiD is active only when part of this trimer. Another possibility is that as more genes are added into the overexpression system, the overall level of each gene product is reduced. This possibility is reflected in the decreased amount of product that can be isolated from the strains as the number of genes increases (see the TLCs in the supplemental material). If CbiD is soluble only when produced under a certain critical concentration and insoluble above that concentration, then it may be soluble only in strain CR727, which bears the most genes and makes the least product.

There is also a conundrum with strain CR727 in that there is no evidence for amidation beyond the formation of cobyrinic acid a,c-diamide even though the strain contains cbiP, the gene for the second amidase. In the aerobic pathway, it has been shown that the substrate for the second amidase (CobQ) is adenosyl-cobyrinic acid a,c-diamide (12). Although E. coli K-12 does have BtuR, an endogenous corrinoid:ATP adenosyltransferase (13), which could serve to adenosylate cobyrinic acid a,c-diamide, it is possible that either the growth conditions or lack of the needed enzymes and/or cofactors prevent the cobalt reduction that is necessary for adenosylation.

Perhaps the most intriguing outcome of this study is that the pathway was able to advance as far as the bisamidated intermediate in the absence of C-1 methylation. Because the order of methylations in the anaerobic pathway has been shown to be as it is depicted in Fig. 1 (21), the substrate specificity of the enzymes ensuing C-1 methylation must be relaxed enough to accept the unmethylated substrates. It is of considerable evolutionary interest to speculate whether the 1-demethyl pathway could be extended to the cobalamin cofactors and, indeed, whether the 1-demethyl cofactors would function in the reactions of the enzymes where they were used.


   FOOTNOTES
 
* This work was supported by National Institutes of Health MERIT Award DK32034 (to A. I. S.) and the Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental material. Back

{ddagger} To whom correspondence should be addressed. Tel.: 979-845-8985; Fax: 979-845-5992; E-Mail: c-roessner{at}tamu.edu.

1 The abbreviations used are: ALA, 5-aminolevulinic acid; cobester, cobyrinic acid heptamethylester. Back


   ACKNOWLEDGMENTS
 
We thank Glenda Crawford for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Roessner, C. A., Santander, P. J., and Scott, A. I. (2001) Vitamins and Hormones (Litwack, G., and Begley, T., eds) Vol. 61, pp. 267–297, Academic Press, San Diego, CA[Medline] [Order article via Infotrieve]
  2. Blanche F., Cameron, B., Crouzet, J., Debussche, L., Thibaut, D., Vuilhorgne, M., Leeper, F. J., and Battersby, A. R. (1995) Angew. Chem. Int. Ed. Engl. 34, 383–411[CrossRef]
  3. Santander, P. J., Roessner, C. A., Stolowich, N. J., Holderman, M. T., and Scott A. I. (1997) Chem. Biol. 4, 659–666[CrossRef][Medline] [Order article via Infotrieve]
  4. Raux, E., Lanois, A., Levillayer, F., Warren, M. J., Brody, E., Rambach, A., and Thermes, C. (1996) J. Bacteriol. 178, 753–767[Abstract/Free Full Text]
  5. Raux, E., Lanois, A., Rambach, A., Warren, M. J., and Thermes, C. (1998) Biochem. J. 335, 167–173
  6. Warren, M. J., Raux, E., Schubert, H., and Escalante-Semerena, J. C. (2002) Nat. Prod. Rep. 19, 390–412[CrossRef][Medline] [Order article via Infotrieve]
  7. Roper, J. M., Raux, E., Brindley, A. A., Schubert, H. L., Gharbia, S. E., Shah, H. N., and Warren, M. J. (2000) J. Biol. Chem. 275, 40316–40323[Abstract/Free Full Text]
  8. Debussche, L., Thibaut, D., Cameron, B., Crouzet, J., and Blanche, F. (1993) J. Bacteriol. 175, 7430–7440[Abstract/Free Full Text]
  9. Bruggemann, H., Henne, A., Hoster, F., Liesegang, H., Wiezer, A., Strittmatter, A., Hujer, S., Durre, P., Gottschalk, G. (2004) Science 305, 671–673[Abstract/Free Full Text]
  10. Roessner, C. A., Warren, M. J., Santander, P. J., Atshaves, B. P., Ozaki, S-I., Stolowich, N. J., Iida, K., and Scott, A. I. (1992) FEBS Lett. 301, 73–78[CrossRef][Medline] [Order article via Infotrieve]
  11. In Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1992) Short Protocols in Molecular Biology, 2nd Ed., John Wiley & Sons, Inc., New York
  12. Blanche, F., Couder, M., Debussche, L., Thibaut, D., Cameron, B., and Crouzet, J. (1991) J. Bacteriol. 173, 6046–6051[Abstract/Free Full Text]
  13. Lundrigan, M. D., and Kadner, R. J. (1989) J. Bacteriol. 171, 154–161[Abstract/Free Full Text]
  14. Yanisch-Perron, Vieira, C. J., and Messing, J. (1985) Gene (Amst.) 33, 103[CrossRef][Medline] [Order article via Infotrieve]
  15. Schreiber, S. L., and Verdine, G. L. (1991) Tetrahedron, 47, 2543–2562[CrossRef]
  16. Guzman, L-M., Belin, D., Carson, M. J., and Beckwith, J. (1995) J. Bacteriol. 177, 4121–4130[Abstract/Free Full Text]
  17. Lutz, R., and Bujard, H. (1997) Nucleic Acids Res. 25, 1203–1210[Abstract/Free Full Text]
  18. Roth, J. R., Lawrence, G. J., Rubenfield, M., Kieffer-Higgens, S., and Church, G. M. (1993) J. Bacteriol. 175, 3303–3316[Abstract/Free Full Text]
  19. Sattler, I., Roessner, C. A., Stolowich, N. J., Hardin, S. H., Harris-Haller, L. W., Yokubaitis, N. T., Murooka, Y., Hashimoto, Y., and Scott, A. I. (1995) J. Bacteriol. 177, 1564–1569[Abstract/Free Full Text]
  20. Battersby, A. R., Edington, C., Fookes, C. J. R., and Hook, J. M. (1982) J. Chem. Soc. Perkin Trans. I 1982, 2265–2272
  21. Scott, A. I., Mackenzie, N. E., Santander, P. J., Fagerness, P. E., Muller, G., Schneider, E., Sedlmeier, R., and Worner, G. (1984) Bioorg. Chem. 12, 356–360[CrossRef]

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
 J. Bacteriol.Home page
C. A. Roessner and A. I. Scott
Fine-Tuning Our Knowledge of the Anaerobic Route to Cobalamin (Vitamin B12)
J. Bacteriol., November 1, 2006; 188(21): 7331 - 7334.
[Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. J. Warren
Finding the final pieces of the vitamin B12 biosynthetic jigsaw
PNAS, March 28, 2006; 103(13): 4799 - 4800.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/17/16748    most recent
M501805200v1
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 Roessner, C. A.
Right arrow Articles by Scott, A. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Roessner, C. A.
Right arrow Articles by Scott, A. I.
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 © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement