Khlebodarova T M, Serov O L
Biokhimiia. 1977 Aug;42(8):1380-6.
An improved method is described for the isolation of isozymes 1 and 5 of lactate dehydrogenase (LDH) from heart and skeletal muscles of foxes. The method includes salt fractionation with ammonium sulphate, chromatography on DEAE- and CM-celluloses and affinity chromatography on AMP-Sepharose. The preparations of LDH isozymes 1 and 5 turned out to be homogeneous both in 7,5% polyacrylamide gel electrophoresis and under immunodiffusion analysis. It is shown that the pH optimum for LDH-1 is 10.2-10.4 for LDH-5 it is 9.5-9.6 in the case of the direct reaction, and the pH optimum is 7.6-7.8 for LDH-1 and 7.3-7.4 for LDH-5 in the case of reverse reaction. The values of Mikhaelis constants were determined for substrates and coenzymes in direct and reverse reactions. It is found that the excess of lactate and pyruvate causes substrate inhibition of both LDH-1 and LDH-5. The activities of LDH-1 and LDH-5 showed an unexpected similar sensitivity to the inhibitory effect of high pyruvate concentrations.
描述了一种从狐狸的心脏和骨骼肌中分离乳酸脱氢酶（LDH）同工酶1和5的改进方法。该方法包括用硫酸铵进行盐分级分离、在DEAE - 纤维素和CM - 纤维素上进行色谱分离以及在AMP - 琼脂糖上进行亲和色谱分离。结果表明，LDH同工酶1和5的制剂在7.5%聚丙烯酰胺凝胶电泳和免疫扩散分析中均为均一的。结果表明，对于直接反应，LDH - 1的最适pH为10.2 - 10.4，LDH - 5为9.5 - 9.6；对于逆反应，LDH - 1的最适pH为7.6 - 7.8，LDH - 5为7.3 - 7.4。测定了直接反应和逆反应中底物和辅酶的米氏常数。发现过量的乳酸和丙酮酸会对LDH - 1和LDH - 5产生底物抑制作用。LDH - 1和LDH - 5的活性对高丙酮酸浓度的抑制作用表现出意外的相似敏感性。
