Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1970 Apr;117(3):601–607. doi: 10.1042/bj1170601

Cathepsin D. Purification of isoenzymes from human and chicken liver

A J Barrett 1
PMCID: PMC1178965  PMID: 5419752

Abstract

1. The Barrett (1967) assay for cathepsin D was slightly modified. 2. The enzyme was purified from liver of man and chicken by a procedure involving autolysis, acetone fractionation, ion-exchange chromatography and isoelectric focusing. 3. Several isoenzymes of cathepsin D were resolved in the isoelectric-focusing step, and three major forms, α,β and γ, were distinguished for each species. 4. A modified analytical method of isoelectric focusing in polyacrylamide gel indicated a high degree of homogeneity of the purified β and γ isoenzymes from each species, and this was supported by their constant high specific activities. 5. Gel filtration of the isoenzymes in a calibrated column of Sephadex G-100 showed that each had a molecular weight of 45000. 6. Human cathepsin D had a pH optimum of 3.5, and that of chicken enzyme was 3.0, haemoglobin being used as substrate. In each species, the three isoenzymes have the same pH-dependence curve. 7. The purified cathepsin D samples showed very little action on acid-denatured albumin.

Full text

PDF
601

Images in this article

604-1PLATE 1
on p.604-1
604-2PLATE 2
on p.604-2
605Fig. 3.
on p.605

Selected References

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

  1. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrett A. J. Cathepsins in pathology: an immunoenzymic study. Biochem J. 1969 Dec;115(5):36P–37P. doi: 10.1042/bj1150036p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrett A. J. Lysosomal acid proteinase of rabbit liver. Biochem J. 1967 Aug;104(2):601–608. doi: 10.1042/bj1040601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barrett A. J., Poole A. R. Unsuitability of leucine naphthylamide for the histochemical demonstration of lysosomal proteolytic activity. Nature. 1969 Oct 18;224(5216):279–280. doi: 10.1038/224279a0. [DOI] [PubMed] [Google Scholar]
  5. Brewer J. M. Artifact produced in disc electrophoresis by ammonium persulfate. Science. 1967 Apr 14;156(3772):256–257. doi: 10.1126/science.156.3772.256. [DOI] [PubMed] [Google Scholar]
  6. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  7. Kress L. F., Peanasky R. J., Klitgaard H. M. Purification, properties, and specificity of hog thyroid proteinase. Biochim Biophys Acta. 1966 Feb 14;113(2):375–389. doi: 10.1016/s0926-6593(66)80076-0. [DOI] [PubMed] [Google Scholar]
  8. LAPRESLE C., WEBB T. The purification and properties of a proteolytic enzyme, rabbit cathepsin E, and further studies on rabbit cathepsin D. Biochem J. 1962 Sep;84:455–462. doi: 10.1042/bj0840455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Lebez D., Turk V., Kregar I. The possibility of differentiation of cathepsins on the basis of their ability to degrade various protein substrates. Enzymologia. 1968 Jul 15;34(5):344–348. [PubMed] [Google Scholar]
  11. Loening U. E. The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem J. 1967 Jan;102(1):251–257. doi: 10.1042/bj1020251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mitchell W. M. A potential source of electrophoretic artifacts in polyacrylamide gels. Biochim Biophys Acta. 1967 Sep 19;147(1):171–174. doi: 10.1016/0005-2795(67)90101-8. [DOI] [PubMed] [Google Scholar]
  13. PRESS E. M., PORTER R. R., CEBRA J. The isolation and properties of a proteolytic enzyme, cathepsin D, from bovine spleen. Biochem J. 1960 Mar;74:501–514. doi: 10.1042/bj0740501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Riley R. F., Coleman M. K. Isoelectric fractionation of proteins on a microscale in polyacrylamide and agarose matrices. J Lab Clin Med. 1968 Oct;72(4):714–720. [PubMed] [Google Scholar]
  15. Schwabe C., Kalnitsky G. A peptidohydrolase from mammalian fibroblasts (bovine dental pulp). Biochemistry. 1966 Jan;5(1):158–168. doi: 10.1021/bi00865a021. [DOI] [PubMed] [Google Scholar]
  16. Smith G. D., Murray M. A., Nichol L. W., Trikojus V. M. Thyroid acid proteinase. Properties and inactivation by diazoacetyl-norleucine methyl ester. Biochim Biophys Acta. 1969 Feb 11;171(2):288–298. doi: 10.1016/0005-2744(69)90162-4. [DOI] [PubMed] [Google Scholar]
  17. TALLAN H. H., JONES M. E., FRUTON J. S. On the proteolytic enzymes of animal tissues. X. Beef spleen cathepsin C. J Biol Chem. 1952 Feb;194(2):793–805. [PubMed] [Google Scholar]
  18. Turk V., Kregar I., Lebez D. Some properties of cathepsin E from bovine spleen. Enzymologia. 1968 Feb 29;34(2):89–100. [PubMed] [Google Scholar]
  19. Weston P. D. A specific antiserum to lysosomal cathepsin D. Immunology. 1969 Sep;17(3):421–428. [PMC free article] [PubMed] [Google Scholar]
  20. Weston P. D., Barrett A. J., Dingle J. T. Specific inhibition of cartilage breakdown. Nature. 1969 Apr 19;222(5190):285–286. doi: 10.1038/222285b0. [DOI] [PubMed] [Google Scholar]
  21. Wrigley C. W. Analytical fractionation of plant and animal proteins by gel electrofocusing. J Chromatogr. 1968 Aug 27;36(3):362–365. doi: 10.1016/s0021-9673(01)92959-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES