Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1983 Dec 1;215(3):519–523. doi: 10.1042/bj2150519

Purification of endopeptidase-24.11 ('enkephalinase') from pig brain by immunoadsorbent chromatography.

J M Relton, N S Gee, R Matsas, A J Turner, A J Kenny
PMCID: PMC1152431  PMID: 6419725

Abstract

Membrane preparations from striatum of pig brain contain endopeptidase activity towards iodoinsulin B-chain. Only 50% of the hydrolysis of insulin B-chain is inhibitable by phosphoramidon, and DEAE-cellulose chromatography can resolve the phosphoramidon-sensitive and -insensitive activities. The former activity (now designated 'endopeptidase-24.11') is responsible for hydrolysis of [D-Ala2,Leu5]enkephalin and is identical with an enzyme in brain that has previously been referred to as 'enkephalinase'. Pig striatal endopeptidase-24.11 has now been purified to homogeneity in a single step by immunoadsorbent chromatography using a monoclonal antibody. The overall purification was 23 000-fold, with a yield of 30%. The brain enzyme appears to be identical with kidney endopeptidase-24.11 in amino acid composition as well as by immunological and kinetic criteria. However, it differs slightly in apparent subunit size (Mr = 87 000), attributable to differences in glycosylation.

Full text

PDF
519

Images in this article

522Fig. 1.
on p.522
522Fig. 2.
on p.522

Selected References

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

  1. Almenoff J., Wilk S., Orlowski M. Membrane bound pituitary metalloendopeptidase: apparent identity to enkephalinase. Biochem Biophys Res Commun. 1981 Sep 16;102(1):206–214. doi: 10.1016/0006-291x(81)91508-4. [DOI] [PubMed] [Google Scholar]
  2. Beynon R. J., Shannon J. D., Bond J. S. Purification and characterization of a metallo-endoproteinase from mouse kidney. Biochem J. 1981 Dec 1;199(3):591–598. doi: 10.1042/bj1990591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chaplin M. F. A rapid and sensitive method for the analysis of carbohydrate components in glycoproteins using gas-liquid chromatography. Anal Biochem. 1982 Jul 1;123(2):336–341. doi: 10.1016/0003-2697(82)90455-9. [DOI] [PubMed] [Google Scholar]
  4. Deschodt-Lanckman M., Strosberg A. D. In vitro degradation of the C-terminal octapeptide of cholecystokinin by 'enkephalinase A'. FEBS Lett. 1983 Feb 7;152(1):109–113. doi: 10.1016/0014-5793(83)80493-1. [DOI] [PubMed] [Google Scholar]
  5. Fulcher I. S., Chaplin M. F., Kenny A. J. Endopeptidase-24.11 purified from pig intestine is differently glycosylated from that in kidney. Biochem J. 1983 Nov 1;215(2):317–323. doi: 10.1042/bj2150317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Gorenstein C., Snyder S. H. Two distinct enkephalinases: solubilization, partial purification and separation from angiotensin converting enzyme. Life Sci. 1979 Dec 10;25(24-25):2065–2070. doi: 10.1016/0024-3205(79)90198-x. [DOI] [PubMed] [Google Scholar]
  9. Kenny A. J., Fulcher I. S. Microvillar endopeptidase, an enzyme with special topological features and a wide distribution. Ciba Found Symp. 1983;95:12–33. doi: 10.1002/9780470720769.ch3. [DOI] [PubMed] [Google Scholar]
  10. Kenny A. J., Fulcher I. S., Ridgwell K., Ingram J. Microvillar membrane neutral endopeptidases. Acta Biol Med Ger. 1981;40(10-11):1465–1471. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Roques B. P., Fournié-Zaluski M. C., Soroca E., Lecomte J. M., Malfroy B., Llorens C., Schwartz J. C. The enkephalinase inhibitor thiorphan shows antinociceptive activity in mice. Nature. 1980 Nov 20;288(5788):286–288. doi: 10.1038/288286a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES