Isolation and Characterization of an Alanyl Aminopeptidase from Rat Liver Cytosol as a Puromycin-Sensitive Enkephalin-Degrading Aminopeptidase
Isolation and Characterization of an Alanyl Aminopeptidase from Rat Liver Cytosol as a Puromycin-Sensitive Enkephalin-Degrading Aminopeptidase
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作为嘌呤霉素敏感脑啡肽降解氨基肽酶，从大鼠肝细胞溶胶中分离并表征丙氨酰氨基肽酶
DOI：
10.1515/bchm.1998.379.6.711
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发表时间：
1998
影响因子：
3.7
通讯作者：
K. Nishi
中科院分区：
文献类型：
--
作者：
Yoshiori Yamamoto;Yao;Kai Huang;I. Ohkubo;K. Nishi
从大鼠肝胞液中纯化了丙氨酰氨肽酶（AAP-S）。在Sephacryl S-200 HR上计算纯化酶的分子量约为100，000，在β-巯基乙醇存在下，在SDS-PAGE上计算纯化酶的分子量约为90，000。这些结果表明，该酶存在于大鼠肝细胞质中的单体形式。在pH7.5 ~ 8.0范围内，该酶能快速水解底物Ala-、Tyr-和Met-MCAs，并适度水解Arg-、Lys-、Leu-、Phe-和Lys-Ala-MCAs。该酶还水解几种氨基酸4-甲基-香豆酰-7-酰胺（MCA）底物。在最适pH（pH7.5）下，AAP-S的k（cat）/Km值大小顺序为：Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs。抑制剂有bestatin、leuhistin、actinonin、amastatin、1，10-phen？roline、PCMBS、Zn ~（2+）、Cd ~（2+）、Co ~（2+）、Cu ~（2+）、Hg ~（2+）和嘌呤霉素。该酶前43位氨基酸序列为Pro 1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro 10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu 20-（Cys）-Leu-Lys-Pro-Asp 25-Leu-Leu- Asp-Phe-Thr 30-Phe-Glu-Gly-Lys-Leu 35-Glu-Ala-Ala-Ala-Gln 40-Val-Arg-Gln-。该N-末端氨基酸序列与大鼠和人脑中嘌呤霉素敏感性脑啡肽降解氨基肽酶以及小鼠神经母细胞瘤细胞系Neuro 2A的N-末端氨基酸序列几乎相同。这些结果表明，AAP-S从大鼠肝细胞质是嘌呤霉素敏感的氨基肽酶。此外，用免疫组织化学的酶强烈染色的大鼠肝细胞的胞质溶胶。
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.
将 130 千道尔顿的人胆汁伴刀豆球蛋白 A 结合蛋白鉴定为氨肽酶 N。
影响因子：
29.4
作者：
Offner,GD;Gong,D;Afdhal,NH
通讯作者：
Afdhal,NH
影响因子：
15.9
作者：
LOOK, AT;ASHMUN, RA;PEIPER, SC
通讯作者：
PEIPER, SC