大豆L-(+)-乳酸脱氢酶：纯化、特性鉴定及亚基结构解析

Soybean L-(+)-lactate dehydrogenases: purification, characterization, and resolution of subunit structure.

作者信息

Tihanyi K, Fontanell A, Talbot B, Thirion J P

机构信息

Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.

出版信息

Arch Biochem Biophys. 1989 Nov 1;274(2):626-32. doi: 10.1016/0003-9861(89)90478-5.

DOI:10.1016/0003-9861(89)90478-5

PMID:2802633

Abstract

Lactate dehydrogenase (LDH) (EC 1.1.1.27) from soybean (Glycine max) was purified 2360-fold to homogeneity using ion-exchange, hydroxyapatite, affinity, and hydrophobic chromatographies. The molecular weight of the holoenzyme is 150,000 +/- 5000. Two-dimensional (isoelectrofocusing-sodium dodecyl sulfate) gel electrophoresis reveals two polypeptides subunits of 5.9 and 6.5 pI and of 36,000 +/- 1000 and 37,000 +/- 1000 Mr, respectively. Nondissociating electrophoresis and isoelectric focusing of LDH resolved five tetrameric isoenzymes with pI's between 6.0 and 6.5. The data suggest that these LDH isoenzymes are derived from random association of the products of two different, but most probably related, genes. Kinetic measurements revealed substrate inhibition at high concentrations of lactic acid and biphasic kinetics with NAD.

摘要

利用离子交换、羟基磷灰石、亲和及疏水色谱法，将来自大豆（ Glycine max ）的乳酸脱氢酶（LDH）（EC 1.1.1.27）纯化了2360倍，达到了均一性。全酶的分子量为150,000±5000。二维（等电聚焦-十二烷基硫酸钠）凝胶电泳显示出两个多肽亚基，其等电点分别为5.9和6.5，分子量分别为36,000±1000和37,000±1000。LDH的非解离电泳和等电聚焦分离出了五种四聚体同工酶，其等电点在6.0至6.5之间。数据表明，这些LDH同工酶源自两个不同但很可能相关的基因产物的随机组合。动力学测量显示，在高浓度乳酸下存在底物抑制，且与NAD呈现双相动力学。