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. 1988 Jul 1;253(1):299–302. doi: 10.1042/bj2530299

A fluorimetric assay for aminopeptidase W.

M C Jackson 1, Y Choudry 1, A Bourne 1, J F Woodley 1, A J Kenny 1
PMCID: PMC1149291  PMID: 3421947

Abstract

A novel two-step enzyme-linked assay for aminopeptidase W is described and validated by comparison with other assays. L-alpha-Glutamyl-L-tryptophan (Glu-Trp) is a favoured substrate for this enzyme. With the use of glutamate dehydrogenase (EC 1.4.1.2) in a second step, the assay measured the release of free glutamate from L-alpha-glutamyl-L-tryptophan by the increase in NADH fluorescence. In the presence of 5 mM-1,10-phenanthroline and 50 microM-cilastatin the contribution of other membrane peptidases, in particular aminopeptidases N and A and microsomal dipeptidase in kidney, was very small. Residual cytosolic activities hydrolysing Glu-Trp were sensitive to inhibition by 2.5 mM-N-ethylmaleimide. The activity of aminopeptidase W was unaffected by these inhibitors. There was good correlation between the fluorimetric assay and those in which the free tryptophan released by kidney membrane fractions was determined by h.p.l.c. or the aminopeptidase W was measured immunoradiometrically with a monoclonal antibody.

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Selected References

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

  1. 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]
  2. Bruce G., Woodley J. F. An accurate fluorometric method to measure the breakdown of gliadin and gliadin peptides. Clin Chim Acta. 1981 Dec 24;117(3):325–332. doi: 10.1016/0009-8981(81)90120-0. [DOI] [PubMed] [Google Scholar]
  3. Campbell B. J., Forrester L. J., Zahler W. L., Burks M. Beta-lactamase activity of purified and partially characterized human renal dipeptidase. J Biol Chem. 1984 Dec 10;259(23):14586–14590. [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. 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]
  6. Gee N. S., Kenny A. J. Proteins of the kidney microvillar membrane. Enzymic and molecular properties of aminopeptidase W. Biochem J. 1987 Aug 15;246(1):97–102. doi: 10.1042/bj2460097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gee N. S., Kenny A. J. Proteins of the kidney microvillar membrane. The 130 kDa protein in pig kidney, recognized by monoclonal antibody GK5C1, is an ectoenzyme with aminopeptidase activity. Biochem J. 1985 Sep 15;230(3):753–764. doi: 10.1042/bj2300753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hirota T., Nishikawa Y., Tanaka M., Igarashi T., Kitagawa H. Characterization of dehydropeptidase I in the rat lung. Eur J Biochem. 1986 Nov 3;160(3):521–525. doi: 10.1111/j.1432-1033.1986.tb10070.x. [DOI] [PubMed] [Google Scholar]
  9. Hooper N. M., Low M. G., Turner A. J. Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C. Biochem J. 1987 Jun 1;244(2):465–469. doi: 10.1042/bj2440465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kessler M., Acuto O., Storelli C., Murer H., Müller M., Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim Biophys Acta. 1978 Jan 4;506(1):136–154. doi: 10.1016/0005-2736(78)90440-6. [DOI] [PubMed] [Google Scholar]
  11. Louvard D., Maroux S., Baratti J., Desnuelle P. On the distribution of enterokinase in porcine intestine and on its subcellular localization. Biochim Biophys Acta. 1973 May 5;309(1):127–137. doi: 10.1016/0005-2744(73)90324-0. [DOI] [PubMed] [Google Scholar]
  12. Matsas R., Stephenson S. L., Hryszko J., Kenny A. J., Turner A. J. The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N. Biochem J. 1985 Oct 15;231(2):445–449. doi: 10.1042/bj2310445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Peterson G. L. Determination of total protein. Methods Enzymol. 1983;91:95–119. doi: 10.1016/s0076-6879(83)91014-5. [DOI] [PubMed] [Google Scholar]

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