Emes A V, Gallimore N J, Hodson A W, Latner A L
Biochem J. 1974 Nov;143(2):453-60. doi: 10.1042/bj1430453.
A method is presented for the preparation of human heart lactate dehydrogenase (l-lactate-NAD(+) oxidoreductase; EC 1.1.1.27) isoenzyme 1; this involves the use of polyacrylamide-gel electrophoresis as a preparative step. The yield was about 10% with a final specific activity of 220 units/mg of protein, one unit being defined as the amount of enzyme catalysing the oxidation of 1mumol of NADH/min at 25 degrees C, in the presence of 0.33mm-pyruvate. The crystalline preparation contained less than 2% of the other isoenzymes, was homogeneous in the ultracentrifuge and showed only a trace of protein contamination on polyacrylamide-gel electrophoresis. Some properties of the crystalline isoenzyme are reported; E(1%) (1cm)=13.2 at 280nm, s(0) (20,w)=7.43S, pI=4.6, and the apparent K(m) for pyruvate=1.02x10(-4)m. The human isoenzyme and the isoenzyme from pig heart differ with respect to amino acid composition, electrophoretic mobility and solubility. It is possible that these differences do not involve the active site, or sites, but are due to changes in amino acid residues elsewhere in the molecule. The importance of purified human LDH-1 isoenzyme with regard to enzyme radioimmunoassay is emphasized.
本文介绍了一种制备人心脏乳酸脱氢酶（L-乳酸-NAD(+)氧化还原酶；EC 1.1.1.27）同工酶1的方法；这涉及使用聚丙烯酰胺凝胶电泳作为制备步骤。产率约为10%，最终比活性为220单位/毫克蛋白质，一个单位定义为在25℃、存在0.33mmol丙酮酸的情况下每分钟催化氧化1μmol NADH的酶量。结晶制剂中其他同工酶含量低于2%，在超速离心机中呈均一性，在聚丙烯酰胺凝胶电泳上仅显示微量蛋白质污染。报道了结晶同工酶的一些性质；在280nm处E(1%) (1cm)=13.2，s(0) (20,w)=7.43S，pI=4.6，丙酮酸的表观K(m)=1.02×10(-4)m。人同工酶和猪心脏同工酶在氨基酸组成、电泳迁移率和溶解度方面存在差异。这些差异可能不涉及活性位点，但可能是由于分子中其他部位氨基酸残基的变化所致。强调了纯化的人LDH-1同工酶在酶放射免疫测定方面的重要性。
