Solubilization and Characterization of Two Rat Brain Membrane-Bound Aminopeptidases Active on Met-Enkephalin

Two aminopeptidases which hydrolyze Metenkephalin at the Tyr-Gly bond have been solubilized from rat brain membranes and resolved by ion-exchange chromatography. These aminopeptidases are designated MI and Mil based on the order in which they are eluted during ion-exchange chromatography. The two aminopeptidases can be distinguished kinetically; aminopeptidase MI hydrolyzes Larginine β-naphthylamide 17 times faster than L-alanine (β-naphthylamide, while only a 1.7-fold difference is exhibited by aminopeptidase MII Aminopeptidase MII exhibits a higher affinity for amino acid β-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 ethylenediaminetetraacetate (EDTA) but can be reconstituted with CoCl₂ and MnCl₂. In contrast, 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 α- and γ-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.