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 liv er cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100000 on Sephacryl S-200 HR and to be 90000 on S DS-PAGE in the presence of P-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 r anging from 7.5 to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat/)K(m) values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala-grea ter than or equal to Leu-greater than or equal to Phe-greater than or equal to Tyr-greater than or equal to Lys-Ala-MCAs. It was strongly in hibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthro line, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined a s -Phe-Glu-Arg-Leu-Pro(10.)-Thr-Glu-Val-Ser-Pro(15)- -Lys-Leu(35)-Glu- Ala-Ala-Ala-Gln(40)-Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycinsensitive enkephalin-degrad ing aminopeptidases in rat and human brains, and the mouse neuroblasto ma cell line Neuro2A, These findings suggest that the AAP-S from rat l iver cytosol is a puromycin-sensitive aminopeptidase. Furthermore, wit h immunohistochemistry the enzyme was strongly stained in the cytosol of the rat liver cells.