Gainey P A, Phelps C F
Biochem J. 1974 Sep;141(3):667-73. doi: 10.1042/bj1410667.
The binding of NAD(+) and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD(+) and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3mum. The binding of NAD(+) to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60-70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.
采用凝胶过滤和荧光滴定法研究了NAD(+)和NADH与牛肝UDP-葡萄糖脱氢酶的结合情况。在底物和产物的饱和浓度下，该酶每摩尔亚基结合0.5摩尔NAD(+)和2摩尔NADH。NADH的解离常数为4.3μm。NAD(+)与酶的结合导致蛋白质荧光有少量淬灭，而NADH的结合导致蛋白质荧光有更大程度（60 - 70%）的淬灭。NADH与酶的结合依赖于pH值。在pH8.1时，用NADH滴定酶得到双相曲线，而在pH8.8时，滴定曲线为双曲线。UDP-木糖，以及程度稍小的UDP-葡萄糖醛酸，降低了酶对NADH的表观亲和力。
