Purification and Characterization of Human Placental Microsomal Aminopeptidase: Immunological Difference between Placental Microsomal Aminopeptidase and Pregnancy Serum Cystyl-Aminopeptidase

Abstract

Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin- Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by highperformance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the K(m) value for this substrate was 1.1 mmol/1. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP).

Cultured human synovial fibroblasts rapidly metabolize kinins and neuropeptides.

“…Regoli and Drapeau, Sherbrooke University, Sherbrooke, Quebec). Purified AmM (AmM) and AmM antisera were prepared by one of us (Dr. Mizutani) as previously described (27).…”

Human Aminopeptidases: A Review of the Literature

“…Human alanine aminopeptidase has been found to be ptesent in virtually all tissues Studieid, with relatively high specific activities in the brush border membranes of kidney proximal tubules and intestine and in bile canalicular membranes (21 -27). Human alanine aminopeptidase has been purified from liver (8, 12, 28 -30), kidney (11,(31)(32)(33), intestine (13,34,35), placenta (31,36) and blood plasma (37,38). Relative molecular mass estimations of the enzyme range from about 150000 (39-41) (obtained by electrophoresis of the non-purified serum enzyme) to about 240 000 for purified enzymes by gel filtration (16,28,42).…”

“…One difference between the two purified preparations was the relatively high alanyl-ß-naphthylamidase acitivity of the placental enzyme preparation. The preferential hydrolysis of leucylarylamides by cystyl aminopeptidase is a general finding (36,38,40,48,56). Some characteristic properties of cystyl aminopeptidase which distinguish it from some other aminopeptidases, are its heat lability and resistance to methionine, bestatin and amastatin inhibition (15,57).…”

“…The substrate specificities are rather broad, and pH optima and the action of effectors are often found to be substrate-or tissue-dependent (55,61,62). It has been shown that the placenta also contains aminopeptidase A (63) and microsomal alanine aminopeptidase, which is immunologically different from serum cystyl aminopeptidase but identical with kidney alanine aminopeptidase (31,36). Several groups have observed a placental enzyme which shares with cystyl aminopeptidase its insensitivity to methionine, its heat lability and the capacity to hydrolyse cystyl-di-ß-naphthylamide, while it has in common with alanine aminopeptidase its microsomal localization and electrophoretic mobility (52,54,58).…”

Physiological Role of Placental Proteases: Interaction Between Pregnancy-Induced Bioactive Peptides and Proteases

“…We purified this enzyme from human placental microsomal fractions [6]. We used immobilized bestatin, the product of streptomyces, which is a powerful inhibitor of aminopeptidases N and B, for the affinity column in the purification of this enzyme [7]. Molecular cloning showed that AP-N is identical to the cell surface cluster of the differentiation antigen (CD13) [8].…”