McLoughlin D J, MacQuarrie R
Biochim Biophys Acta. 1978 Nov 10;527(1):204-11. doi: 10.1016/0005-2744(78)90269-3.
The major isozymic form of sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from rabbit liver using a simplified three-step chromatographic procedure involving an ion exchange and two affinity chromatography steps. The 1200-fold purified enzyme is electrophoretically homogeneous, nucleotide-free, and possesses a specific activity of 295 units/mg and an isoelectric point of 6.5. A steady-state kinetic analysis was applied to both the forward and reverse reactions. The NADH oxidation reaction was found to adhere to Michaelis-Menten behavior with Km values of 22 micrometer and 75 micrometer for NADH and dihydroxyacetone phosphate, respectively. In the NAD reduction reaction, sigmoidal kinetic patterns were observed when NAD was the variable substrate whereas with sn-glycerol-3-phosphate as the variable substrate, strictly hyperbolic kinetics were observed. The apparent Km values for NAD and glycerol-3-phosphate were 83 and 909 micrometer, respectively. By comparison with published reports, these results demonstrate that the rabbit muscle and liver isozymes of sn-glycerol-3-phosphate dehydrogenase have different kinetic properties and suggest that the liver isozyme is better adapted to participation in glyconeogenesis in vivo.
已使用一种简化的三步色谱法从兔肝中纯化出sn-甘油-3-磷酸脱氢酶（sn-甘油-3-磷酸：NAD+ 2-氧化还原酶，EC 1.1.1.8）的主要同工酶形式，该方法包括一个离子交换步骤和两个亲和色谱步骤。经过1200倍纯化的酶在电泳上是均一的，不含核苷酸，比活性为295单位/毫克，等电点为6.5。对正向和反向反应都进行了稳态动力学分析。发现NADH氧化反应符合米氏行为，NADH和磷酸二羟丙酮的Km值分别为22微摩尔和75微摩尔。在NAD还原反应中，当NAD为可变底物时观察到S形动力学模式，而当以sn-甘油-3-磷酸作为可变底物时，观察到严格的双曲线动力学。NAD和甘油-3-磷酸的表观Km值分别为83和909微摩尔。与已发表的报告相比，这些结果表明兔肌肉和肝脏中的sn-甘油-3-磷酸脱氢酶同工酶具有不同的动力学性质，并表明肝脏同工酶更适合于体内参与糖异生作用。
