SUBCELLULAR LOCALIZATION AND PARTIAL PURIFICATION OF A CHLORIDE DEPENDENT ANGIOTENSIN-I CONVERTING ENZYME FROM RAT BRAIN

Abstract— Angiotensin converting enzyme (peptidyl dipeptide hydrolase EC 3.4.15.1) was extracted from particulates of rat brain using the nonionic detergent Triton X-100. Enzyme activity in subcellular fractions was associated with purified synaptosomes and present in the microsomal fraction, but absent in purified mitochondria and water-shocked myelin. Partial purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite columns. The enzyme had a pH optimum of pH 7–8 and an apparent K_m of 2.2 m m using hippuryl-histidyl-leucine as substrate; it was chloride dependent, inhibited by (Sar¹-Ala⁸)-angiotensin-II (saralasin), and, at lower concentrations, by the specific nonapeptide inhibitor SQ 20881. Associated with the purified enzyme was an aminopeptidase, cleaving N-terminal Asp from the native substrate, which could be involved in the production of the active heptapeptide, angiotensin III (des-Asp-angiotensin-II). Also present was a carboxypeptidase-like enzyme removing C-terminal Phe following the liberation of His-Leu by converting enzyme, which may be involved in the inactivation of angiotensin II or III.