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. 1987 Feb 15;242(1):281–283. doi: 10.1042/bj2420281

An improved spectrophotometric assay for leucine aminopeptidase.

R E Beattie, D J Guthrie, D T Elmore, C H Williams, B Walker
PMCID: PMC1147694  PMID: 3593241

Abstract

A sensitive assay to determine the activity of leucine aminopeptidase (EC 3.4.11.1), using L-leucine thiobenzyl ester as substrate, was developed. Hydrolysis of the ester by leucine aminopeptidase can be monitored in the presence of 5,5-dithiobis-(2-nitrobenzoic acid) by continuous spectrophotometric measurement at 412 nm. Comparison with some amide substrates showed that the thiol ester provides a much more sensitive assay, its specificity constant (Vmax./Km) being some 3000-fold higher than that of leucine p-nitroanilide.

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Selected References

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  1. BINKLEY F., TORRES C. Spectrophotometric assay of peptidase activity. Arch Biochem Biophys. 1960 Feb;86:201–203. doi: 10.1016/0003-9861(60)90404-5. [DOI] [PubMed] [Google Scholar]
  2. Castillo M. J., Nakajima K., Zimmerman M., Powers J. C. Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases. Anal Biochem. 1979 Oct 15;99(1):53–64. doi: 10.1016/0003-2697(79)90043-5. [DOI] [PubMed] [Google Scholar]
  3. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  4. Farmer D. A., Hageman J. H. Use of N-benzoyl-L-tyrosine thiobenzyl ester as a protease substrate. Hydrolysis by alpha-chymotrypsin and subtilisin BPN. J Biol Chem. 1975 Sep 25;250(18):7366–7371. [PubMed] [Google Scholar]
  5. GREENBERG L. J. Fluorometric measurement of alkaline phosphatase and aminopeptidase activities in the order of 10-14 mole. Biochem Biophys Res Commun. 1962 Nov 27;9:430–435. doi: 10.1016/0006-291x(62)90029-3. [DOI] [PubMed] [Google Scholar]
  6. GREEN M. N., TSOU K. C., BRESSLER R., SELIGMAN A. M. The colorimetric determination of leucine aminopeptidase activity with L-leucyl-beta-naphthylamide hydrochloride. Arch Biochem Biophys. 1955 Aug;57(2):458–474. doi: 10.1016/0003-9861(55)90307-6. [DOI] [PubMed] [Google Scholar]
  7. Green G. D., Shaw E. Thiobenzyl benzyloxycarbonyl-L-lysinate, substrate for a sensitive colorimetric assay for trypsin-like enzymes. Anal Biochem. 1979 Mar;93(2):223–226. doi: 10.1016/s0003-2697(79)80141-4. [DOI] [PubMed] [Google Scholar]
  8. Hanson H., Lasch J. Enzymatische Transamidierungsreaktion mit kristalliner Rinderaugenlinsen-Leucinaminopeptidase (EC 3.4.1.1) Hoppe Seylers Z Physiol Chem. 1967 Nov;348(11):1525–1539. [PubMed] [Google Scholar]
  9. Henson H., Frohne M. Crystalline leucine aminopeptidase from lens (alpha-aminoacyl-peptide hydrolase; EC 3.4.11.1). Methods Enzymol. 1976;45:504–520. doi: 10.1016/s0076-6879(76)45045-0. [DOI] [PubMed] [Google Scholar]
  10. Hwang S. Y., Kingsbury W. D., Hall N. M., Jakas D. R., Dunn G. L., Gilvarg C. Determination of leucine aminopeptidase using phenylalanyl-3-thia-phenylalanine as substrate. Anal Biochem. 1986 May 1;154(2):552–558. doi: 10.1016/0003-2697(86)90028-x. [DOI] [PubMed] [Google Scholar]
  11. Lee H. J., LaRue J. N., Wilson I. B. A simple spectrophotometric assay for amino acyl arylamidases (naphthylamidases, aminopeptidases). Anal Biochem. 1971 Jun;41(2):397–401. doi: 10.1016/0003-2697(71)90157-6. [DOI] [PubMed] [Google Scholar]
  12. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  13. McRae B. J., Kurachi K., Heimark R. L., Fujikawa K., Davie E. W., Powers J. C. Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates. Biochemistry. 1981 Dec 8;20(25):7196–7206. doi: 10.1021/bi00528a022. [DOI] [PubMed] [Google Scholar]
  14. Saifuku K., Sekine T., Namihisa T., Takahashi T., Kanaoka Y. A novel fluorometric ultramicro determination of serum leucine aminopeptidase using a coumarine derivative. Clin Chim Acta. 1978 Mar 1;84(1-2):85–91. doi: 10.1016/0009-8981(78)90479-5. [DOI] [PubMed] [Google Scholar]
  15. Shen C., Mellius P. Leucine aminopeptidase from swine kidney: purification, molecular weight, subunit and amino acid composition. Prep Biochem. 1977;7(3-4):243–256. doi: 10.1080/00327487708061641. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Van Wart H. E., Lin S. H. Metal binding stoichiometry and mechanism of metal ion modulation of the activity of porcine kidney leucine aminopeptidase. Biochemistry. 1981 Sep 29;20(20):5682–5689. doi: 10.1021/bi00523a007. [DOI] [PubMed] [Google Scholar]
  18. WEYGAND F., NINTZ E. WEIERE PEPTIDSYNTHESEN MIT PMZ-AMINOSAEUREN. Z Naturforsch B. 1965 May;20:429–434. [PubMed] [Google Scholar]
  19. Yasutake A., Powers J. C. Reactivity of human leukocyte elastase and porcine pancreatic elastase toward peptide 4-nitroanilides containing model desmosine residues. Evidence that human leukocyte elastase is selective for cross-linked regions of elastin. Biochemistry. 1981 Jun 23;20(13):3675–3679. doi: 10.1021/bi00516a002. [DOI] [PubMed] [Google Scholar]
  20. Zimmerman M., Yurewicz E., Patel G. A new fluorogenic substrate for chymotrypsin. Anal Biochem. 1976 Jan;70(1):258–262. doi: 10.1016/s0003-2697(76)80066-8. [DOI] [PubMed] [Google Scholar]

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