Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization.
A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.
Yamamoto
Y
Y
Department of Legal Medicine, Shiga University of Medical Science, Otsu, 520-2192, Shiga, Japan.
Li
Y H
YH
Ushiyama
I
I
Nishimura
A
A
Ohkubo
I
I
Nishi
K
K
D000626:Aminopeptidases / Q000737:chemistry; D000818:Animals; D018826:CD13 Antigens / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011500:Protein Synthesis Inhibitors; D011691:Puromycin; D051381:Rats; D017386:Sequence Homology, Amino Acid
D011500:Protein Synthesis Inhibitors; D011691:Puromycin; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme; D018826:CD13 Antigens