来自朗氏锥虫的α-甘露糖苷酶的纯化及部分特性分析

Purification and partial characterization of alpha-mannosidase from Trypanosoma rangeli.

作者信息

Nok A J, Shuaibu M N, Kanbara H, Yanagi T

机构信息

Protozoology Department, Institute of Tropical Medicine, Nagasaki University, Japan.

出版信息

Parasitol Res. 2000 Nov;86(11):923-8. doi: 10.1007/s004360000274.

DOI:10.1007/s004360000274

PMID:11097301

Abstract

An alpha-mannosidase from Trypanosoma rangeli was partially purified by a protocol involving solubilization using lysis buffer followed by chromatography on diethylaminoethyl (DEAE)-cellulose and Sephadex 100 columns. The enzyme has a molecular weight of 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and of 90 kDa as determined by gel filtration. The purified T. rangeli alpha-mannosidase has a pH optimum ranging between 5 and 6 and a temperature optimum of 37 degrees C. It has activation energy of 8.15 x 10(2) J mol(-1) K(-1). The enzyme has a Michaelis constant (Km) of 99 microM for the 4-methylumbelliferyl-alpha-D-mannopyranoside substrate (MU-alpha-mann). It is strongly inhibited by swainsonine [inhibition constant (Ki) 0.048 microM] and is moderately inhibited by mannose and alpha-D-methylmannopyranoside. The enzyme activity is decreased in the presence of 1 mM Ca2+, Co2+, Cu2+, Fe2+, Fe3+, Hg2+, Mg2+, and Mn2+.

摘要

通过一种方法对来自兰氏锥虫的α-甘露糖苷酶进行了部分纯化，该方法包括使用裂解缓冲液溶解，随后在二乙氨基乙基（DEAE）-纤维素和葡聚糖100柱上进行层析。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，该酶的分子量为45 kDa，通过凝胶过滤测定为90 kDa。纯化后的兰氏锥虫α-甘露糖苷酶的最适pH值在5至6之间，最适温度为37℃。其活化能为8.15×10² J mol⁻¹ K⁻¹。该酶对4-甲基伞形酮基-α-D-甘露吡喃糖苷底物（MU-α-甘露糖）的米氏常数（Km）为99 μM。它受到苦马豆素的强烈抑制[抑制常数（Ki）为0.048 μM]，并受到甘露糖和α-D-甲基甘露吡喃糖苷的中度抑制。在1 mM Ca²⁺、Co²⁺、Cu²⁺、Fe²⁺、Fe³⁺、Hg²⁺、Mg²⁺和Mn²⁺存在的情况下，酶活性降低。