Doughty M J
Department of Vision Science, Glasgow-Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, Scotland.
Exp Eye Res. 1998 Feb;66(2):231-9. doi: 10.1006/exer.1997.0423.
The purpose of the following study was to examine the kinetic properties of lactate dehydrogenase (LDH) in cell extracts of corneal epithelium. The epithelium, from quality- and size-selected ewe corneas, was dispersed in 10 mM Trizma maleate with or without sucrose at pH 7.0 and cell fractions obtained by homogenisation and differential centrifugation for evaluation of LDH at 37 degrees C. Pyruvate-dependent oxidation of NADH was optimal at pH 6.5, while lactate-dependent reduction of NAD was optimal at pH 9.5; the former activity averaged 3 to 3.5 fold higher than the latter. NADPH and NADP were poor cofactors. The apparent Km values for pyruvate and lactate were 99 micromoles and 3.9 millimoles respectively. At pH 6. 5, substrate inhibition was observed with pyruvate in excess of 0.25 mm with 50% activity measured at 1.2 mm. Excess substrate effects were not seen with lactate at pH 9.5, but pyruvate inhibited lactate-dependent reduction of NAD with 50% activity measured at 1.1 mm. Differential centrifugation confirmed that the activity was predominantly localised in the soluble (post mitochondria-lysosome) fraction of the cells.
以下研究的目的是检测角膜上皮细胞提取物中乳酸脱氢酶（LDH）的动力学特性。从质量和大小筛选后的母羊角膜获取上皮组织，将其分散于pH 7.0、含或不含蔗糖的10 mM马来酸三羟甲基氨基甲烷中，经匀浆和差速离心获得细胞组分，以评估37℃时的LDH。丙酮酸依赖的NADH氧化在pH 6.5时最佳，而乳酸依赖的NAD还原在pH 9.5时最佳；前者活性平均比后者高3至3.5倍。NADPH和NADP是较差的辅助因子。丙酮酸和乳酸的表观Km值分别为99微摩尔和3.9毫摩尔。在pH 6.5时，当丙酮酸浓度超过0.25 mM时会观察到底物抑制，在1.2 mM时测得50%的活性。在pH 9.5时，乳酸未出现过量底物效应，但丙酮酸会抑制乳酸依赖的NAD还原，在1.1 mM时测得50%的活性。差速离心证实该活性主要定位于细胞的可溶性（线粒体 - 溶酶体后）组分中。
