Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Jul 15;221(2):445–452. doi: 10.1042/bj2210445

Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

A D Gounaris, M A Brown, A J Barrett
PMCID: PMC1144057  PMID: 6548132

Abstract

Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.

Full text

PDF

Images in this article

449Fig. 2.
on p.449
450Fig. 3.
on p.450
451Fig. 4.
on p.451

Selected References

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

  1. Anastasi A., Brown M. A., Kembhavi A. A., Nicklin M. J., Sayers C. A., Sunter D. C., Barrett A. J. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J. 1983 Apr 1;211(1):129–138. doi: 10.1042/bj2110129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baines B. S., Brocklehurst K. A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods. Biochem J. 1979 Feb 1;177(2):541–548. doi: 10.1042/bj1770541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrett A. J., Brown M. A., Sayers C. A. The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule. Biochem J. 1979 Aug 1;181(2):401–418. doi: 10.1042/bj1810401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barrett A. J. Chicken alpha2-proteinase inhibitor: a serum protein homologous with ovoinhibitor of egg white. Biochim Biophys Acta. 1974 Nov 5;371(1):52–62. doi: 10.1016/0005-2795(74)90154-8. [DOI] [PubMed] [Google Scholar]
  5. Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M., Hanada K. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189–198. doi: 10.1042/bj2010189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
  7. Green G. D., Kembhavi A. A., Davies M. E., Barrett A. J. Cystatin-like cysteine proteinase inhibitors from human liver. Biochem J. 1984 Mar 15;218(3):939–946. doi: 10.1042/bj2180939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Henderson P. J. A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem J. 1972 Apr;127(2):321–333. doi: 10.1042/bj1270321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Howell J. B., Beck T., Bates B., Hunter M. J. Interaction of alpha 2-macroglobulin with trypsin, chymotrypsin, plasmin, and papain. Arch Biochem Biophys. 1983 Feb 15;221(1):261–270. doi: 10.1016/0003-9861(83)90143-1. [DOI] [PubMed] [Google Scholar]
  10. Järvinen M. Purification and some characteristics of two human serum proteins inhibiting papain and other thiol proteinases. FEBS Lett. 1979 Dec 15;108(2):461–464. doi: 10.1016/0014-5793(79)80588-8. [DOI] [PubMed] [Google Scholar]
  11. Laskowski M., Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem. 1980;49:593–626. doi: 10.1146/annurev.bi.49.070180.003113. [DOI] [PubMed] [Google Scholar]
  12. Nagase H., Barrett A. J. Human plasma kallikrein. A rapid purification method with high yield. Biochem J. 1981 Jan 1;193(1):187–192. doi: 10.1042/bj1930187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ryley H. C. Isolation and partial characterisation of a thiol proteinase inhibitor from human plasma. Biochem Biophys Res Commun. 1979 Aug 13;89(3):871–878. doi: 10.1016/0006-291x(79)91859-x. [DOI] [PubMed] [Google Scholar]
  14. Sasaki M., Minakata K., Yamamoto H., Niwa M., Kato T., Ito N. A new serum component which specifically inhibits thiol proteinases. Biochem Biophys Res Commun. 1977 Jun 6;76(3):917–924. doi: 10.1016/0006-291x(77)91589-3. [DOI] [PubMed] [Google Scholar]
  15. Sasaki M., Taniguchi K., Minakata K. Multimolecular forms of thiol proteinase inhibitor in human plasma. J Biochem. 1981 Jan;89(1):169–177. doi: 10.1093/oxfordjournals.jbchem.a133178. [DOI] [PubMed] [Google Scholar]
  16. Sasaki M., Taniguchi K., Suzuki K., Imahori K. Human plasma alpha 1- and alpha 2-thiol proteinase inhibitors strongly inhibit Ca-activated neutral protease from muscle. Biochem Biophys Res Commun. 1983 Jan 14;110(1):256–261. doi: 10.1016/0006-291x(83)91288-3. [DOI] [PubMed] [Google Scholar]
  17. Schwartz W. N., Barrett A. J. Human cathepsin H. Biochem J. 1980 Nov 1;191(2):487–497. doi: 10.1042/bj1910487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Starkey P. M., Barrett A. J. Inhibition by alpha-macroglobulin and other serum proteins. Biochem J. 1973 Apr;131(4):823–831. doi: 10.1042/bj1310823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Taniguchi K., Ito J., Sasaki M. Partial purification and properties of urinary thiol proteinase inhibitors. J Biochem. 1981 Jan;89(1):179–184. doi: 10.1093/oxfordjournals.jbchem.a133179. [DOI] [PubMed] [Google Scholar]
  20. Travis J., Salvesen G. S. Human plasma proteinase inhibitors. Annu Rev Biochem. 1983;52:655–709. doi: 10.1146/annurev.bi.52.070183.003255. [DOI] [PubMed] [Google Scholar]
  21. Valeri A. M., Wilson S. M., Feinman R. D. Reaction of antithrombin with proteases. Evidence for a specific reaction with papain. Biochim Biophys Acta. 1980 Aug 7;614(2):526–533. doi: 10.1016/0005-2744(80)90241-7. [DOI] [PubMed] [Google Scholar]
  22. WILKINSON G. N. Statistical estimations in enzyme kinetics. Biochem J. 1961 Aug;80:324–332. doi: 10.1042/bj0800324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yoshida N., Weksler B., Nachman R. Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function. J Biol Chem. 1983 Jun 10;258(11):7168–7174. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES