Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Mar 14;92(6):2189–2193. doi: 10.1073/pnas.92.6.2189

Glycine reductase selenoprotein A is not a glycoprotein: the positive periodic acid-Schiff reagent test is the result of peptide bond cleavage and carbonyl group generation.

Y Kimura 1, T C Stadtman 1
PMCID: PMC42449  PMID: 7892245

Abstract

The complete amino acid sequence of Clostridium sticklandii selenoprotein A, a selenocysteine-containing protein component of the glycine reductase complex, has been established. Both the intact protein and peptide fragments produced by Staphylococcus aureus V8 protease or trypsin were purified by reversed-phase high-performance liquid chromatography and subjected to electrospray ionization mass spectrometric analysis and standard Edman degradation. Selenoprotein A consists of 157 amino acids with a chemical molecular weight of 17,011, in reasonable agreement with the observed molecular weight (17,022.7) determined from its ionization mass spectrum. The sequence of the amino-terminal region of the isolated native protein is Ser-Arg-Phe-Thr-Gly-Lys- Lys-Ile-Val-Ile-Ile-Gly-Asp-Arg-Asp-. An N-terminal methionine residue deduced from the gene sequence was not present. Although selenoprotein A reacted positively in a glycoprotein stain when using either the periodic acid-Schiff reagent procedure or a commercial glycan detection kit, no saccharide was detected by carbohydrate analyses after acid hydrolysis or methanolysis. Identity of the amino acid sequence determined by analysis with that deduced from the gene sequence is further evidence of the absence of bound carbohydrate.

Full text

PDF
2189

Images in this article

2190Fig. 1
on p.2190

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cone J. E., Del Río R. M., Davis J. N., Stadtman T. C. Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2659–2663. doi: 10.1073/pnas.73.8.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cone J. E., del Río R. M., Stadtman T. C. Clostridial glycine reductase complex. Purification and characterization of the selenoprotein component. J Biol Chem. 1977 Aug 10;252(15):5337–5344. [PubMed] [Google Scholar]
  3. Dietrichs D., Meyer M., Rieth M., Andreesen J. R. Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]. J Bacteriol. 1991 Oct;173(19):5983–5991. doi: 10.1128/jb.173.19.5983-5991.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  5. Garcia G. E., Stadtman T. C. Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing, and expression in Escherichia coli. J Bacteriol. 1992 Nov;174(22):7080–7089. doi: 10.1128/jb.174.22.7080-7089.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Garcia G. E., Stadtman T. C. Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA. J Bacteriol. 1991 Mar;173(6):2093–2098. doi: 10.1128/jb.173.6.2093-2098.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gerwig G. J., Kamerling J. P., Vliegenthart J. F., Morag E., Lamed R., Bayer E. A. The nature of the carbohydrate-peptide linkage region in glycoproteins from the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens. J Biol Chem. 1993 Dec 25;268(36):26956–26960. [PubMed] [Google Scholar]
  8. Heinrikson R. L., Meredith S. C. Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem. 1984 Jan;136(1):65–74. doi: 10.1016/0003-2697(84)90307-5. [DOI] [PubMed] [Google Scholar]
  9. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lenz A. G., Costabel U., Shaltiel S., Levine R. L. Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride. Anal Biochem. 1989 Mar;177(2):419–425. doi: 10.1016/0003-2697(89)90077-8. [DOI] [PubMed] [Google Scholar]
  12. Messner P., Christian R., Kolbe J., Schulz G., Sleytr U. B. Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70. J Bacteriol. 1992 Apr;174(7):2236–2240. doi: 10.1128/jb.174.7.2236-2240.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Messner P., Sleytr U. B. Bacterial surface layer glycoproteins. Glycobiology. 1991 Dec;1(6):545–551. doi: 10.1093/glycob/1.6.545. [DOI] [PubMed] [Google Scholar]
  14. Sliwkowski M. X., Stadtman T. C. Purification and immunological studies of selenoprotein A of the clostridial glycine reductase complex. J Biol Chem. 1987 Apr 5;262(10):4899–4904. [PubMed] [Google Scholar]
  15. Sliwkowski M. X., Stadtman T. C. Selenium-dependent glycine reductase: differences in physicochemical properties and biological activities of selenoprotein A components isolated from Clostridium sticklandii and Clostridium purinolyticum. Biofactors. 1988 Dec;1(4):293–296. [PubMed] [Google Scholar]
  16. Sliwkowski M. X., Stadtman T. C. Selenoprotein A of the clostridial glycine reductase complex: purification and amino acid sequence of the selenocysteine-containing peptide. Proc Natl Acad Sci U S A. 1988 Jan;85(2):368–371. doi: 10.1073/pnas.85.2.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stadtman T. C. Glycine reduction to acetate and ammonia: identification of ferredoxin and another low molecular weight acidic protein as components of the reductase system. Arch Biochem Biophys. 1966 Jan;113(1):9–19. doi: 10.1016/0003-9861(66)90151-2. [DOI] [PubMed] [Google Scholar]
  18. Turner D. C., Stadtman T. C. Purification of protein components of the clostridial glycine reductase system and characterization of protein A as a selenoprotein. Arch Biochem Biophys. 1973 Jan;154(1):366–381. doi: 10.1016/0003-9861(73)90069-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES