A specific enzyme assay for aminopeptidase M in rat brain

Terrence J. Gillespie; Pierre N.M. Konings; Bradley J. Merrill; Thomas P. Davis

doi:10.1016/0024-3205(92)90161-H

A specific enzyme assay for aminopeptidase M in rat brain

Terrence J. Gillespie, Pierre N.M. Konings, Bradley J. Merrill, Thomas P. Davis

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

Abstract

A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 μM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 μM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 μM) had only a slight effect on the K_m of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The K_m of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the K_m of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.

Original language	English (US)
Pages (from-to)	2097-2106
Number of pages	10
Journal	Life Sciences
Volume	51
Issue number	26
DOIs	https://doi.org/10.1016/0024-3205(92)90161-H
State	Published - 1992

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology
General Pharmacology, Toxicology and Pharmaceutics

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title = "A specific enzyme assay for aminopeptidase M in rat brain",

abstract = "A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 μM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 μM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 μM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.",

author = "Gillespie, {Terrence J.} and Konings, {Pierre N.M.} and Merrill, {Bradley J.} and Davis, {Thomas P.}",

year = "1992",

doi = "10.1016/0024-3205(92)90161-H",

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pages = "2097--2106",

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T1 - A specific enzyme assay for aminopeptidase M in rat brain

AU - Gillespie, Terrence J.

AU - Konings, Pierre N.M.

AU - Merrill, Bradley J.

AU - Davis, Thomas P.

PY - 1992

Y1 - 1992

N2 - A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 μM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 μM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 μM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.

AB - A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 μM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 μM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 μM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.

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A specific enzyme assay for aminopeptidase M in rat brain

Abstract

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