Immunoaffinity purification and characterization of native placental leucine aminopeptidase/oxytocinase from human placenta

cDNA cloning of placental leucine aminopeptidase (P-LAP)/cystinyl aminopept idase (CAP)/oxytocinase demonstrated that this enzyme is a type II integral membrane protein, which means that native P-LAP, found in placenta, is mem brane-bound and that the soluble form of this enzyme, found in maternal ser a, is most likely derived from the native form. The presence of the differe nt forms of the protein makes it difficult to purify homogeneously. In the current study we prepared antibody specific to native P-LAP and used it to purify native P-LAP from microsomal fractions of human placenta to homogene ity, 5039-fold within 4 h, by immunoaffinity chromatography. Zn2+ and Cu2strongly inhibited the enzyme but Ca2+ did not. Amastatin was a more potent inhibitor than bestatin and leupeptin. Using antibodies against native P-L AP, protein having 83 per cent of L-methionine insensitive Leu-p-nitroanili de cleaving activity, was immunoprecipitated from the microsomal fraction o f human placenta. The availability of a specific antibody against native P-LAP permits the ra pid purification and the preliminary immunoassay of the enzyme. Establishme nt of simple purification and assay methods for the native, membrane bound form of P-LAP pave the way to elucidating the roles and processing systems of this enzyme. (C) 2000 Harcourt Publishers Ltd.