Purification and properties of human intestine alanine aminopeptidase.
Human intestinal alanine aminopeptidase has been purified to greater than 90% homogeneity. The enzyme was released from mucosal cell membranes by Triton X-100 treatment. The native enzyme had a molecular weight of 206,000 in dilute buffer and 108,000 in the presence of sodium dodecyl sulfate. The enzyme was inhibited by chelators suggesting the presence of a metal ion in the enzyme. The most potent chelator inhibitor tested, o-phenanthroline, gave mixed kinetics (Ki = 67 micro M). Activity was restored by removal of the chelator. The enzyme was inhibited competitively by amino acids having hydrophobic side chains such as L-phenylalanine (Ki = 0.67 mM). Puromycin and methicillin also inhibited the enzyme in the competitive (Ki = 12.5 micro M) and noncompetitive (Ki = 4.6 mM) manner, respectively. Kinetic analysis of several amino acid beta-naphthylamides as substrates demonstrated the preference for substrates having hydrophobic or basic amino terminal residues with no beta-branching. L-Methionyl-beta-naphthylamide was the most tightly bound with L-alanyl-beta-naphthylamide was the most rapidly hydrolyzed.
D000409:Alanine / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D018826:CD13 Antigens; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D006358:Hot Temperature; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008712:Methicillin / Q000494:pharmacology; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D010649:Phenylalanine / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D013379:Substrate Specificity
D002614:Chelating Agents; D010649:Phenylalanine; D011691:Puromycin; D009439:Neuraminidase; D000626:Aminopeptidases; D018826:CD13 Antigens; D000409:Alanine; D008712:Methicillin