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. 1990 May 1;267(3):665–669. doi: 10.1042/bj2670665

Substrate and inhibitor studies with human gastric aspartic proteinases.

A Baxter 1, C J Campbell 1, C J Grinham 1, R M Keane 1, B C Lawton 1, J E Pendlebury 1
PMCID: PMC1131349  PMID: 2111133

Abstract

The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.

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Selected References

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

  1. Bell A. E., Sellers L. A., Allen A., Cunliffe W. J., Morris E. R., Ross-Murphy S. B. Properties of gastric and duodenal mucus: effect of proteolysis, disulfide reduction, bile, acid, ethanol, and hypertonicity on mucus gel structure. Gastroenterology. 1985 Jan;88(1 Pt 2):269–280. doi: 10.1016/s0016-5085(85)80180-3. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Dunn B. M., Jimenez M., Parten B. F., Valler M. J., Rolph C. E., Kay J. A systematic series of synthetic chromophoric substrates for aspartic proteinases. Biochem J. 1986 Aug 1;237(3):899–906. doi: 10.1042/bj2370899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dunn B. M., Valler M. J., Rolph C. E., Foundling S. I., Jimenez M., Kay J. The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2. Biochim Biophys Acta. 1987 Jun 17;913(2):122–130. doi: 10.1016/0167-4838(87)90320-7. [DOI] [PubMed] [Google Scholar]
  5. Foltmann B. Gastric proteinases--structure, function, evolution and mechanism of action. Essays Biochem. 1981;17:52–84. [PubMed] [Google Scholar]
  6. Hille M. B., Barrett A. J., Dingle J. T., Fell H. B. Microassay for cathepsin D shows an unexpected effedt of cycloheximide on limb-bone rudiments in organ culture. Exp Cell Res. 1970 Aug;61(2):470–472. doi: 10.1016/0014-4827(70)90476-3. [DOI] [PubMed] [Google Scholar]
  7. Holladay M. W., Salituro F. G., Rich D. H. Synthetic and enzyme inhibition studies of pepstatin analogues containing hydroxyethylene and ketomethylene dipeptide isosteres. J Med Chem. 1987 Feb;30(2):374–383. doi: 10.1021/jm00385a020. [DOI] [PubMed] [Google Scholar]
  8. Jentoft N., Dearborn D. G. Labeling of proteins by reductive methylation using sodium cyanoborohydride. J Biol Chem. 1979 Jun 10;254(11):4359–4365. [PubMed] [Google Scholar]
  9. Kucerová Z., Pohl J., Korbová L. Separation of human pepsin and gastricsin by affinity chromatography with an immobilized synthetic inhibitor. J Chromatogr. 1986 Apr 11;376:409–412. doi: 10.1016/s0378-4347(00)80857-9. [DOI] [PubMed] [Google Scholar]
  10. Maibaum J., Rich D. H. Inhibition of porcine pepsin by two substrate analogues containing statine. The effect of histidine at the P2 subsite on the inhibition of aspartic proteinases. J Med Chem. 1988 Mar;31(3):625–629. doi: 10.1021/jm00398a022. [DOI] [PubMed] [Google Scholar]
  11. Myren J., Berild D., Orstavik I., Serck Hanssen A. Virus antibodies in the serum of patients with liver disease. Scand J Gastroenterol. 1982 Jan;17(1):5–9. doi: 10.3109/00365528209181035. [DOI] [PubMed] [Google Scholar]
  12. Newton C. J., Roberts N. B., Taylor W. H. Separation of human pepsins in gastric juice by high-performance ion-exchange chromatography. J Chromatogr. 1987 Jul 3;417(2):391–396. doi: 10.1016/0378-4347(87)80134-2. [DOI] [PubMed] [Google Scholar]
  13. Pearson J. P., Ward R., Allen A., Roberts N. B., Taylor W. H. Mucus degradation by pepsin: comparison of mucolytic activity of human pepsin 1 and pepsin 3: implications in peptic ulceration. Gut. 1986 Mar;27(3):243–248. doi: 10.1136/gut.27.3.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pohl J., Dunn B. M. Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site. Biochemistry. 1988 Jun 28;27(13):4827–4834. doi: 10.1021/bi00413a037. [DOI] [PubMed] [Google Scholar]
  15. Rich D. H., Bernatowicz M. S. Synthesis of analogues of the carboxyl protease inhibitor pepstatin. Effect of structure in subsite P3 on inhibition of pepsin. J Med Chem. 1982 Jul;25(7):791–795. doi: 10.1021/jm00349a005. [DOI] [PubMed] [Google Scholar]
  16. Rich D. H., Salituro F. G. Synthesis of analogues of pepstatin. Effect of structure in subsites P1', P2', and P2 on inhibition of porcine pepsin. J Med Chem. 1983 Jun;26(6):904–910. doi: 10.1021/jm00360a022. [DOI] [PubMed] [Google Scholar]
  17. Roberts N. B., Taylor W. H. The preparation and purification of individual human pepsins by using diethylaminoethyl-cellulose. Biochem J. 1978 Mar 1;169(3):607–615. doi: 10.1042/bj1690607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Samloff I. M. Peptic ulcer: the many proteinases of aggression. Gastroenterology. 1989 Feb;96(2 Pt 2 Suppl):586–595. doi: 10.1016/s0016-5085(89)80054-x. [DOI] [PubMed] [Google Scholar]
  19. Samloff I. M., Taggart R. T., Shiraishi T., Branch T., Reid W. A., Heath R., Lewis R. W., Valler M. J., Kay J. Slow moving proteinase. Isolation, characterization, and immunohistochemical localization in gastric mucosa. Gastroenterology. 1987 Jul;93(1):77–84. [PubMed] [Google Scholar]
  20. Scawen M., Allen A. The action of proteolytic enzymes on the glycoprotein from pig gastric mucus. Biochem J. 1977 May 1;163(2):363–368. doi: 10.1042/bj1630363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Valler M. J., Kay J., Aoyagi T., Dunn B. M. The interaction of aspartic proteinases with naturally-occurring inhibitors from actinomycetes and Ascaris lumbricoides. J Enzyme Inhib. 1985;1(1):77–82. doi: 10.3109/14756368509031284. [DOI] [PubMed] [Google Scholar]

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