Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1977 Jun 1;163(3):565–570. doi: 10.1042/bj1630565

Separation and properties of two arylamidases from rat cardiac-muscle extracts.

A F Bury, T Coolbear, C R Savery
PMCID: PMC1164737  PMID: 406901

Abstract

Two main arylamidase activities were separated from a particle-free supernatant of rat heart by chromatography on DEAE-Sephadex. Although both enzymes hydrolysed L-leucine 4-nitroanilide, only peak-II enzyme hydrolysed L-lysine 4-nitroanilide. A third minor peak (Ia) contained an enzyme that was active mainly on the L-lysine 4-nitroanilide. The mol.wts. of the enzymes in peaks I and II were approx. 257000 and 105000 respectively. The pH optimum was approx. pH7.0 for peak-I enzyme and 7.0-8.0 for peak-II enzyme. Both enzymes were inhibited by addition of puromycin, p-hydroxymercuribenzoate, o-phenanthroline and bivalent metal ions. Addition of dithiothreitol resulted in stimulation of both activities. Dialysis against o-phenanthroline resulted in inhibition of peak-I and -II enzymes, but after dialysis against EDTA only peak-II enzyme was inhibited.

Full text

PDF
565

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. Bury A. F., Pennington R. J. Hydrolysis of diepptide 2-naphthylamides by human muscle enzymes. Biochem J. 1975 Feb;145(2):413–416. doi: 10.1042/bj1450413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. ELLIS S. A THIOL-ACTIVATED AMINOPEPTIDASE OF THE PITUITARY. Biochem Biophys Res Commun. 1963 Aug 20;12:452–456. doi: 10.1016/0006-291x(63)90314-0. [DOI] [PubMed] [Google Scholar]
  4. Ellis S., Perry M. Pituitary arylamidases and peptidases. J Biol Chem. 1966 Aug 25;241(16):3679–3686. [PubMed] [Google Scholar]
  5. Femfert U., Pfleiderer G. The effect of cations and complexing agents on the hydrolysis of L-Alanine-4-nitroanilide by aminopeptidase M. FEBS Lett. 1971 Apr;14(2):89–91. doi: 10.1016/0014-5793(71)80107-2. [DOI] [PubMed] [Google Scholar]
  6. Hopsu-Havu V. K., Ekfors T. O. Distribution of a dipeptide naphthylamidase in rat tissues and its localisation by using diazo coupling and labeled antibody techniques. Histochemie. 1969;17(1):30–38. doi: 10.1007/BF00306327. [DOI] [PubMed] [Google Scholar]
  7. Marks N., Datta R. K., Lajtha A. Partial resolution of brain arylamidases and aminopeptidases. J Biol Chem. 1968 Jun 10;243(11):2882–2889. [PubMed] [Google Scholar]
  8. Melius P., Moseley M. H., Brown D. M. Characterization of the subunits of swine kidney leucine aminopeptidase. Biochim Biophys Acta. 1970 Oct 20;221(1):62–68. doi: 10.1016/0005-2795(70)90197-2. [DOI] [PubMed] [Google Scholar]
  9. Mäkinen K. K. Inhibition of arylaminopeptidases and proline iminopeptidases of human whole saliva by benzethonium chloride (benzyldimethyl [2-[2-(p-1,1,3,3-tetramethylbutylphenoxy)ethoxy]-ethyl] ammonium chloride). FEBS Lett. 1968 Dec;2(2):101–104. doi: 10.1016/0014-5793(68)80113-9. [DOI] [PubMed] [Google Scholar]
  10. Mäkinen K. K., Mäkinen P. L. Evidence on erythrocyte aminopeptidase B. Int J Protein Res. 1971;3(1):41–47. doi: 10.1111/j.1399-3011.1971.tb01691.x. [DOI] [PubMed] [Google Scholar]
  11. PATTERSON E. K., HSIAO S. H., KEPPEL A., SOROF S. STUDIES ON DIPEPTIDASES AND AMINOPEPTIDASES. II. ZONAL ELECTROPHORETIC SEPARATION OF RAT LIVER PEPTIDASES. J Biol Chem. 1965 Feb;240:710–716. [PubMed] [Google Scholar]
  12. PATTERSON E. K., HSIAO S. H., KEPPEL A. STUDIES ON DIPEPTIDASES AND AMINOPEPTIDASES. I. DISTINCTION BETWEEN LEUCINE AMINOPEPTIDASE AND ENZYMES THAT HYDROLYZE L-LEUCYL-BETA-NAPHTHYLAMIDE. J Biol Chem. 1963 Nov;238:3611–3620. [PubMed] [Google Scholar]
  13. Parsons M. E., Pennington R. J. Separation of rat muscle aminopeptidases. Biochem J. 1976 May 1;155(2):375–381. doi: 10.1042/bj1550375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rehfeld N., Peters J. E., Giesecke H., Beier L., Haschen R. J. Untersuchungen über Aminosäure-arylamidasen. I. Verteilung und Isoenzyme der Aminosäure-arylamidase im menschlichen Organismus. Acta Biol Med Ger. 1967;19(6):809–818. [PubMed] [Google Scholar]
  15. SMITH E. L., SPACKMAN D. H. Leucine aminopeptidase. V. Activation, specificity, and mechanism of action. J Biol Chem. 1955 Jan;212(1):271–299. [PubMed] [Google Scholar]
  16. Schwabe C. Peptide hydrolases in mammalian connective tissue. II. Leucine aminopeptidase. Purification and evidence for subunit structure. Biochemistry. 1969 Mar;8(3):783–794. doi: 10.1021/bi00831a005. [DOI] [PubMed] [Google Scholar]
  17. Suszkiw J. B., Brecher A. S. Brain aminoacyl arylamidase. Further purification of the soluble bovine enzyme and studies on substrate specificity and possible active-site residues. Biochemistry. 1970 Sep 29;9(20):4008–4017. doi: 10.1021/bi00822a021. [DOI] [PubMed] [Google Scholar]
  18. Szewczuk A., Kochman M., Baranowski T. Dipeptide nitriles as substrates for colorimetric determination of aminopeptidases. Acta Biochim Pol. 1965;12(4):357–367. [PubMed] [Google Scholar]
  19. TUPPY H., WIESBAUER U., WINTERSBERGER E. [Amino acid-p-nitroanilide as a substrate for aminopeptidases and other proteolytic enzymes]. Hoppe Seylers Z Physiol Chem. 1962 Nov 15;329:278–288. doi: 10.1515/bchm2.1962.329.1.278. [DOI] [PubMed] [Google Scholar]
  20. Wachsmuth E. D., Fritze I., Pfleiderer G. An aminopeptidase occurring in pig kidney. II. A study on the mechanism of the hydrolysis. Biochemistry. 1966 Jan;5(1):175–182. doi: 10.1021/bi00865a023. [DOI] [PubMed] [Google Scholar]
  21. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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

RESOURCES