Williams R A, Andrews P
Biochem J. 1986 Jun 15;236(3):721-7. doi: 10.1042/bj2360721.
The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo.
乳房链球菌N.C.D.O. 2039细胞中的1,6-二磷酸果糖[Fru(1,6)P2]依赖性乳酸脱氢酶通过一种程序进行纯化，该程序包括在磷酸盐缓冲液中于DEAE-纤维素和蓝色琼脂糖CL-6B上进行色谱分离。该酶似乎通过NADH结合位点与蓝色琼脂糖相互作用。纯化后的酶具有的催化特性通常与其他Fru(1,6)P2依赖性乳酸脱氢酶相似，并且在没有Fru(1,6)P2时没有催化活性。通过超速离心、分析凝胶过滤和活性测量研究了其在不同条件下以不同形式的存在情况。它由分子量为35,900±500的亚基组成，在存在足够浓度的Fru(1,6)P2、磷酸盐或NADH时，它以四聚体形式存在，而当这些配体浓度较低或不存在时，亚基处于浓度依赖性的缔合-解离平衡状态。解离过程缓慢且使酶失活，尽管添加配体可逆转解离，但丧失的活性最多只能部分恢复。当解离是由温度降低引起时会出现例外情况，此时在原始温度下丧失的活性可完全恢复。四聚体在某些配体浓度下也会在不解离的情况下丧失活性。这些结果共同表明该酶上存在两类Fru(1,6)P2和NADH的结合位点，并且磷酸盐与Fru(1,6)P2结合在相同位点的可能性较大。两种不同的配体一起在防止失活和解离方面比它们单独存在时的效果预期要有效得多。得出的结论是，该酶的四聚体形式而非处于缔合-解离平衡状态的酶参与了其体内活性的调节。
