Banerjee Nupur, Bhattacharyya Debasish
Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mallick Road, Jadavpur, Kolkata 700 032, India.
Indian J Biochem Biophys. 2012 Jun;49(3):182-8.
While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45 degrees C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.
在尝试于pH 8.0条件下从鲤鱼肝脏提取物中纯化UDP-半乳糖4-表异构酶时，观察到即使经过透析，该制剂仍能将NAD还原为NADH，从而干扰表异构酶的测定。在一系列色谱步骤中，NAD还原活性和表异构酶共洗脱。对半纯化级分的质谱分析表明，鲤鱼肝脏乳酸脱氢酶（LDH）含有结合的乳酸，在NAD存在下会转化为丙酮酸。与无底物的LDH相比，酶结合的乳酸以及与表异构酶的结合分别使LDH在胰蛋白酶消化和45℃热失活时稳定了2.7倍和4.2倍。LDH和表异构酶不属于任何一条途径，而是碳水化合物代谢两条不同途径的限速酶。通常，紧密相关的酶协同作用，例如同一代谢途径的两种酶。在此背景下，本研究中重新加载的LDH和表异构酶的共纯化是一种不寻常的现象。
