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 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.