Solubilization and characterization of two rat brain membrane-bound aminopeptidases active on Met-enkephalin

Abstract

Two aminopeptidases which hydrolyze Met-enkephalin at the Tyr-Gly bond were solubilized from rat brain membranes and resolved by ion-exchange chromatography. These aminopeptidases are designated MI and MII based on the order in which they are eluted during ion-exchange chromatography. The 2 aminopeptidases can be distinguished kinetically; aminopeptidase MI hydrolyzes L-arginine .beta.-naphthylamide 17 times faster than L-alanine .beta.-naphthylamide, while only a 1.7-fold difference is exhibited by aminopeptidase MII. Aminopeptidase MII exhibits a higher affinity for amino acid .beta.-naphthylamides, Met-enkephalin, Leu-enkephalin and the inhibitor puromycin as compared to aminopeptidase MI. Greater than 90% of aminopeptidase MII activity is lost upon dialysis against EDTA but can be reconstituted with CoCl2 and MnCl2. Aminopeptidase MI loses only 30% of its activity when dialyzed against EDTA. In addition to cleaving the Tyr-Gly bond of Met-enkephalin, aminopeptidase MII also cleaves the Tyr-Gly bond of .alpha.- and .gamma.-endorphin. Hydrolysis of Met-enkephalin by intact membranes derived from whole rat brain occurs primarily by cleavage at the Tyr-Gly bond, with this activity attributable to aminopeptidase MII.