Purification and Properties of Human Placental Aminopeptidase B

Abstract

Aminopeptidase B (EC 3.4.11.6; L-arginyl-β-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-β-naphthylamide as substrate and the K(m) value for this enzyme was 0.3 mmol/1. Human placental aminopeptidase B was markedly activity by CL. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and purymycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.

Purification and characterization of aminopeptidase B from goat brain

“…The calculated molecular weight of arginyl aminopeptidase from human brain [11] and rat brain cortex [25] is 74 kDa and 60 kDa respectively. The molecular weight of this enzyme from other sources ranged from 43 kDa [4] to 220 kDa [33].…”

Molecular Cloning and Expression of Rat Liver Aminopeptidase B

“…The enzyme hydrolyzed only Arg-and Lys-NA (activity ratio, 100:45), and the activity was inhibited by 1 mM bestatin, o-phenanthroline, and p-chloromercuribenzoic acid, which chemicals are known as inhibitors of Ap-B. The specific activity for hydrolysis of arginyl-NA, 14.3 units/mg of protein, was higher than the values for human (17), rat (4), and porcine (8) Ap-B reported previously.…”

“…Purification of Enzyme from Human Placenta-The purification procedure was developed from that reported by Nagata et al (17), with the following modification. Human placenta was obtained at normal fullterm delivery.…”

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

“…We purified this enzyme from human placenta [26]. Although its involvement in the formation of BK from kallidin-10 was suggested, this enzyme does not seem to be involved in the metabolism of kinins and A-III [26].…”

“…Although its involvement in the formation of BK from kallidin-10 was suggested, this enzyme does not seem to be involved in the metabolism of kinins and A-III [26]. The natural substrate of this enzyme is still unknown.…”