Isolation and characterization of an alanyl aminopeptidase from rat liver cytosol as a puromycin-sensitive enkephalin-degrading aminopeptidase.
Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat liver cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytosol of the rat liver cells.
Department of Legal Medicine, Shiga University of Medical Science, Seta, Otsu, Japan.
Y
Y
Yamamoto
Y H
YH
Li
K
K
Huang
I
I
Ohkubo
K
K
Nishi
D000595:Amino Acid Sequence; D000818:Animals; D018826:CD13 Antigens / Q000302:isolation & purification* / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D004745:Enkephalins / Q000378:metabolism*; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011500:Protein Synthesis Inhibitors / Q000378:metabolism*; D011691:Puromycin / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D004745:Enkephalins; D011500:Protein Synthesis Inhibitors; D011691:Puromycin; D018826:CD13 Antigens