1887

Abstract

in common with several members of the group constitutively produced a number of protein and peptide hydrolysing enzymes. Amongst the most active was an arylamidase, which specifically hydrolysed the dipeptidyl chromogenic substrates glycylprolyl -nitroanilide (GPRPNA), glycylprolyl -naphthylamide (GPNA) and valylalanine -nitroanilide (VAPNA), and had some proteolytic activity towards azocasein. No activity was detected against proline -naphthylamide, glycine, valanine or alanine -nitroanilides. Physiological studies showed that the enzyme was largely cell-associated during exponential growth in batch culture, but was progressively released by the bacteria before the cells entered stationary phase. Glycylprolyl arylamidase (GPA) was completely cell-bound during growth in continuous culture, where synthesis increased concomitantly with dilution rate (specific growth rate) in both carbon- and nitrogen-limited chemostats. Gel-filtration chromatography of cell extracts yielded a single peak of GPA activity, with an apparent molecular mass of . 160 kDa, while one peak of enzyme activity was eluted by 0.3 M NaCl during cation-exchange chromatography. Activity staining of SDS polyacrylamide gels showed a single GPA band at 80 kDa, suggesting that the enzyme was a dimer. Two fractions of GPA activity were recorded during preparative isoelectric focusing with apparent isoelectric points of pH 3.51 (fraction 3) and 3.95 (fraction 6), indicating the possible existence of GPA isoenzymes. GPRPNA, VAPNA and azocasein were hydrolysed by the major fraction (fraction 3), while only the -nitroanilide substrates were hydrolysed by fraction 6. Studies with the partially purified enzyme obtained from gel filtration columns showed a relatively broad pH optimum at 7.5-8.2. Inhibition experiments demonstrated that while aspartic (pepstatin A), thiol (iodoacetate) and metalloprotease (EDTA, cysteine) inhibitors had little effect on hydrolysis of glycylproline -nitroanilide, GPA was strongly inhibited (. 80%) by 5 m phenyl-methylsulphonyl fluoride (PMSF), indicating it to be a serine enzyme.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/00222615-46-7-547
1997-07-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jmm/46/7/medmicro-46-7-547.html?itemId=/content/journal/jmm/10.1099/00222615-46-7-547&mimeType=html&fmt=ahah

References

  1. Holdeman L. V., Cato E. R., Moore W. E. C. Anaerobe laboratory manual. 4th edn Blacksburg: Virginia Polytechnic Institute and State University; 1977
    [Google Scholar]
  2. Finegold S. M., Sutter V. L., Mathisen G. E. Normal indigenous intestinal flora. In Hentges D. J. (ed) Human intestinal microflora in health and disease London: Academic Press; 19833
    [Google Scholar]
  3. Jousimies-Somer H. R., Finegold S. M. Anaerobic Gram-negative bacilli and cocci. In Balows A., Hausler W. J., Herrmann K. L., Isenberg H. D., Shadomy H. J. (eds) Manual of clinical microbiology 5th edn Washington, DC: American Society for Microbiology; 1991538–553
    [Google Scholar]
  4. Macfarlane G. T., Allison C., Gibson S. A. W., Cummings J. H. Contribution of the microflora to proteolysis in the human large intestine. J Appl Bacteriol 1988; 64:37–46
    [Google Scholar]
  5. Macfarlane G. T., Gibson G. R. Characteristics of protease synthesis in Bacteroides splanchnicus NCTC 10825. Appl Microbiol Biotechnol 1993; 39:506–511
    [Google Scholar]
  6. Macfarlane G. T., Macfarlane S. Utilization of pancreatic trypsin and chymotrypsin by proteolytic and nonproteolytic Bacteroides fragilis-type bacteria. Curr Microbiol 1991; 23:143–148
    [Google Scholar]
  7. Macfarlane G. T., Cummings J. H., Macfarlane S., Gibson G. R. Influence of retention time on degradation of pancreatic enzymes by human colonic bacteria grown in a 3-stage continuous culture system. J Appl Bacteriol 1989; 67:520–527
    [Google Scholar]
  8. Macfarlane S., Macfarlane G. T. Proteolysis and amino acid fermentation. In Gibson G. R., Macfarlane G. T. (eds) Human colonic bacteria: role in nutrition, physiology and pathology Boca Raton: CRC Press; 199575
    [Google Scholar]
  9. Macfarlane G. T., Cummings J. H., Allison C. Protein degradation by human intestinal bacteria. J Gen Microbiol 1986; 132:1647–1656
    [Google Scholar]
  10. Gibson S. A. W., Macfarlane G. T. Characterization of proteases formed by Bacteroides fragilis. J Gen Microbiol 1988; 134:2231–2240
    [Google Scholar]
  11. Donelli G., Fabbri A., Fiorentini C. Bacteroides fragilis enterotoxin induces cytoskeletal changes and surface blebbing in HT-29 cells. Infect Immun 1996; 64:113–119
    [Google Scholar]
  12. Riepe S. P., Goldstein J., Alpers D. H. Effect of secreted Bacteroides proteases on human intestinal brush border hydrolases. J Clin Invest 1980; 66:314–322
    [Google Scholar]
  13. Gibson S. A. W., Macfarlane G. T. Studies on the proteolytic activity of Bacteroides fragilis. J Gen Microbiol 1988; 134:19–27
    [Google Scholar]
  14. Macfarlane G. T., Englyst H. N. Starch utilization by the human large intestinal microflora. J Appl Bacteriol 1986; 60:195–201
    [Google Scholar]
  15. Gibson S. A. W., McFarlan C., Hay S., Macfarlane G. T. Significance of microflora in proteolysis in the colon. Appl Environ Microbiol 1989; 55:679–683
    [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685
    [Google Scholar]
  17. Degnan B. A., Macfarlane G. T. Arabinogalactan utilization in continuous cultures of Bifidobacterium longum. Effect of coculture with Bacteroides thetaiotaomicron. Anaerobe 1995; 1:103–112
    [Google Scholar]
  18. Hazlewood G. P., Edwards R. Proteolytic activities of a rumen bacterium, Bacteroides ruminicola R8/4. J Gen Microbiol 1981; 125:11–15
    [Google Scholar]
  19. Umezawa H., Aoyagi T. Activities of proteinase inhibitors of microbial origin. In Barrett A. J. (ed) Proteinases in mammalian cells and tissues Amsterdam: North Holland; 1977637
    [Google Scholar]
  20. Barrett A. J. Introduction to the history and classification of tissue proteinases. In Barrett A. J. (ed) Proteinases in mammalian cells and tissues Amsterdam: North Holland; 19771
    [Google Scholar]
  21. Matsubara H., Feder J. Other bacterial, mold and yeast proteases. In Boyer P. D. (ed) The enzymes vol 3 New York: Academic Press; 1971721
    [Google Scholar]
  22. Siefter S., Harper E. The collagénases. In Boyer P. D. (ed) The enzymes vol 3 New York: Academic Press; 1971649
    [Google Scholar]
  23. Webb J. L. Mercurials. In Enzyme and metabolic inhibitors vol 2 New York: Academic Press; 1966729–985
    [Google Scholar]
  24. Liu P. V., Hsieh H.-C. Inhibition of protease production in various bacteria by ammonium salts: its effect on toxin production and virulence. J Bacteriol 1969; 99:406–413
    [Google Scholar]
  25. Macfarlane G. T., Macfarlane S., Gibson G. R. Synthesis and release of proteases by Bacteroides fragilis. Curr Microbiol 1992; 24:55–60
    [Google Scholar]
  26. Fukasawa K. M., Harada M. Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811. Arch Biochem Biophys 1981; 210:230–237
    [Google Scholar]
  27. Abiko Y., Hayakawa M., Murai S., Takiguchi H. Glycylprolyl dipeptidylaminopeptidase from Bacteroides gingivalis. J Dent Res 1985; 64:106–111
    [Google Scholar]
  28. Macfarlane G. T., Allison C., Gibson G. R. Effect of pH on protease activities in the large intestine. Lett Appl Microbiol 1988; 7:161–164
    [Google Scholar]
  29. Cummings J. H., Pômare E. W., Branch W. J., Naylor C. P. E., Macfarlane G. T. Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut 1987; 28:1221–1227
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/00222615-46-7-547
Loading
/content/journal/jmm/10.1099/00222615-46-7-547
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error