Chiou T K, Matsui T, Konosu S
Laboratory of Marine Biochemistry, Faculty of Agriculture, University of Tokyo.
J Biochem. 1989 Apr;105(4):505-9. doi: 10.1093/oxfordjournals.jbchem.a122696.
An aminopeptidase was isolated from a soluble fraction of Alaska pollack roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The molecular weight of the enzyme was estimated to be 125,000 and 105,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The pH optimum and temperature optimum were 7.2 and 35 degrees C, respectively. The purified enzyme hydrolyzed various alpha-aminoacyl beta-naphthylamides and cleaved L-Ala-beta-naphthylamide most rapidly. Both a sulfhydryl group and a divalent metal ion are essential for activity; however, the enzyme was inhibited when incubated with divalent metal ions. Puromycin, chelating agents, and thiol reagents were effective inhibitors. The enzyme was also inhibited by L-amino acids, in particular glutamic acid. Thus, the Alaska pollack roe aminopeptidase resembles soluble alanyl aminopeptidase [EC 3.4.11.14].
在2-巯基乙醇存在的情况下，通过硫酸铵分级分离以及在DEAE-纤维素、羟基磷灰石和葡聚糖G-200上进行柱色谱，从阿拉斯加狭鳕鱼籽的可溶部分中分离出一种氨肽酶。通过凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分别估计该酶的分子量为125,000和105,000。最适pH和最适温度分别为7.2和35℃。纯化后的酶可水解各种α-氨基酰基β-萘酰胺，并且对L-丙氨酰-β-萘酰胺的切割速度最快。一个巯基和一个二价金属离子对活性至关重要；然而，该酶在与二价金属离子一起孵育时会受到抑制。嘌呤霉素、螯合剂和硫醇试剂是有效的抑制剂。该酶也受到L-氨基酸的抑制，尤其是谷氨酸。因此，阿拉斯加狭鳕鱼籽氨肽酶类似于可溶性丙氨酰氨肽酶[EC 3.4.11.14]。
