An Enkephalin-Degrading Aminopeptidase from Rat Brain Catalyzes the Hydrolysis of a Neuropeptide, Kyotorphin (L-Tyr-L-Arg)

Abstract

We studied the hydrolysis of a neuropeptide kyotorphin (L-Tyr-L-Arg) by an enkephalin-degrading aminopeptidase purified from cytosol of rat brain in vitro. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), gel filtration and isoelectric focusing. The aminopeptidase with an apparent molecular weight (M_r)=98000 catalyzed the hydrolysis of Leu-and Met-enkephalins with K_m values of 125 and 142μM, respectively. The enzyme activity was inhibited by bestatin, amastatin and puromycin but not by pepstatin, leupeptin and phenylmethanesulfonyl fluoride (PMSF). Kyotorphin was degraded by the aminopeptidase at pH 7.0, and the V_max and K_m values were 9.2 μmol/min/mg protein and 95μM, respectively. The K_m value for kyotorphin was compatible to those for Leu- and Met-enkephalins. Taken together, these results suggest a possible involvement of the enkephalin-degrading aminopeptidase in cytosolic degradation of kyotorphin in neuronal cells of rat brain.