Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

Abstract

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP and Sephadex G-200 columns. The overall purification was .apprx. 1200-fold, with 25% yield. The purified enzyme showed 1 band on disc gel electrophoresis and 2 bands on sodium dodecyl sulfate electrophoresis with MW of 62,000 and 66,000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 .mu.mol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze .gamma.- or .beta.-endorphin or dynorpin, but it does hydrolyze neutral and basic aminoacyl .beta.-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum and MW, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M and the soluble aminopeptidase for enkephalin degradation.