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. 1985 Jul 1;229(1):251–257. doi: 10.1042/bj2290251

Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys.

S Hedeager-Sørensen, A J Kenny
PMCID: PMC1145174  PMID: 4038259

Abstract

Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at -20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.

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Selected References

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

  1. Auricchio S., Greco L., de Vizia B., Buonocore V. Dipeptidylaminopeptidase and carboxypeptidase activities of the brush border of rabbit small intestine. Gastroenterology. 1978 Dec;75(6):1073–1079. [PubMed] [Google Scholar]
  2. Booth A. G., Hubbard L. M., Kenny A. J. Proteins of the kidney microvillar membrane. Immunoelectrophoretic analysis of the membrane hydrolases: identification and resolution of the detergent- and proteinase-solubilized forms. Biochem J. 1979 May 1;179(2):397–405. doi: 10.1042/bj1790397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Booth A. G., Kenny A. J. A rapid method for the preparation of microvilli from rabbit kidney. Biochem J. 1974 Sep;142(3):575–581. doi: 10.1042/bj1420575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Danielsen E. M., Norén O., Sjöström H., Ingram J., Kenny A. J. Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms. Biochem J. 1980 Sep 1;189(3):591–603. doi: 10.1042/bj1890591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Das M., Soffer R. L. Pulmonary angiotensin-converting enzyme. Structural and catalytic properties. J Biol Chem. 1975 Sep 10;250(17):6762–6768. [PubMed] [Google Scholar]
  6. Dehm P., Nordwig A. The cleavage of prolyl peptides by kidney peptidases. Isolation of a microsomal carboxypeptidase from swine kidney. Eur J Biochem. 1970 Dec;17(2):372–377. doi: 10.1111/j.1432-1033.1970.tb01175.x. [DOI] [PubMed] [Google Scholar]
  7. Dehm P., Nordwig A. The cleavage of prolyl peptides by kidney peptidases. Partial purification of an "X-prolyl-aminopeptidase" from swine kidney microsomes. Eur J Biochem. 1970 Dec;17(2):364–371. doi: 10.1111/j.1432-1033.1970.tb01174.x. [DOI] [PubMed] [Google Scholar]
  8. Fulcher I. S., Kenny A. J. Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys. Biochem J. 1983 Jun 1;211(3):743–753. doi: 10.1042/bj2110743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gee N. S., Matsas R., Kenny A. J. A monoclonal antibody to kidney endopeptidase-24.11. Its application in immunoadsorbent purification of the enzyme and immunofluorescent microscopy of kidney and intestine. Biochem J. 1983 Aug 15;214(2):377–386. doi: 10.1042/bj2140377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kakimoto T., Oshima G., Yeh H. S., Erdös E. G. Purification of lysosomal prolycarboxypeptidase angiotensinase C. Biochim Biophys Acta. 1973 Mar 15;302(1):178–182. doi: 10.1016/0005-2744(73)90021-1. [DOI] [PubMed] [Google Scholar]
  11. Kenny A. J., Booth A. G., George S. G., Ingram J., Kershaw D., Wood E. J., Young A. R. Dipeptidyl peptidase IV, a kidney brush-border serine peptidase. Biochem J. 1976 Jul 1;157(1):169–182. doi: 10.1042/bj1570169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kenny A. J., Booth A. G., Macnair R. D. Peptidases of the kidney microvillus membrane. Acta Biol Med Ger. 1977;36(11-12):1575–1585. [PubMed] [Google Scholar]
  13. Kenny A. J., Maroux S. Topology of microvillar membrance hydrolases of kidney and intestine. Physiol Rev. 1982 Jan;62(1):91–128. doi: 10.1152/physrev.1982.62.1.91. [DOI] [PubMed] [Google Scholar]
  14. Kerr M. A., Kenny A. J. The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border. Biochem J. 1974 Mar;137(3):489–495. doi: 10.1042/bj1370489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lehky P., Lisowski J., Wolf D. P., Wacker H., Stein E. A. Pig kidney particulate aminopeptidase. A zinc metalloenzyme. Biochim Biophys Acta. 1973 Sep 15;321(1):274–281. doi: 10.1016/0005-2744(73)90082-x. [DOI] [PubMed] [Google Scholar]
  16. Macnair D. C., Kenny A. J. Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV. Biochem J. 1979 May 1;179(2):379–395. doi: 10.1042/bj1790379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matsas R., Fulcher I. S., Kenny A. J., Turner A. J. Substance P and [Leu]enkephalin are hydrolyzed by an enzyme in pig caudate synaptic membranes that is identical with the endopeptidase of kidney microvilli. Proc Natl Acad Sci U S A. 1983 May;80(10):3111–3115. doi: 10.1073/pnas.80.10.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Odya C. E., Erdös E. G. Human prolylcarboxypeptidase. Methods Enzymol. 1981;80(Pt 100):460–466. doi: 10.1016/s0076-6879(81)80040-7. [DOI] [PubMed] [Google Scholar]
  19. Taylor S. L., Tappel A. L. Identification and separation of lysosomal carboxypeptidases. Biochim Biophys Acta. 1974 Mar 21;341(1):99–111. doi: 10.1016/0005-2744(74)90070-9. [DOI] [PubMed] [Google Scholar]
  20. Walter R., Simmons W. H., Yoshimoto T. Proline specific endo- and exopeptidases. Mol Cell Biochem. 1980 Apr 18;30(2):111–127. doi: 10.1007/BF00227927. [DOI] [PubMed] [Google Scholar]
  21. Yang H. Y., Erdös E. G., Chiang T. S. New enzymatic route for the inactivation of angiotensin. Nature. 1968 Jun 29;218(5148):1224–1226. doi: 10.1038/2181224a0. [DOI] [PubMed] [Google Scholar]

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