Jensen J
Institute of Physiology, University of Aarhus, Denmark.
Biochim Biophys Acta. 1992 Sep 21;1110(1):81-7. doi: 10.1016/0005-2736(92)90297-y.
A centrifugation method has been used for determination of [14C]ADP and [3H]ouabain binding to Na,K-ATPase from pig kidney with high specific activity. In the presence of K+, the fit of the [14C]ADP binding data to a two-site model gives a component with high affinity which accounts for 12 +/- 2% of the total sites. The figure is significantly different from 50%, i.e., two components of equal size cannot be assumed. This contrasts with a ratio between the sites of 1:1 obtained by the rate dialysis technique. The discrepancy may be due to the fact that the centrifugation method enables bound ADP to be determined at lower concentrations of free ligand. [3H]Ouabain binding in the absence of Na+ is compatible with a straight line in a Scatchard plot if the isotope is purified shortly before use. An unspecific binding of ouabain can be neglected if the concentration of free ouabain is not too high. In the presence of Na+, the isotherms become upward concave. An analysis of the binding data gives a 19:81% division, although equilibrium is not quite attained. This is a maximum value because the lack in equilibrium will be most pronounced at the small values of free ouabain. Thus the ADP-binding studies are supported. The finding here is in some agreement with the semiquantitative immunoassay showing that pig kidney enzyme contains the isoenzymes alpha 1, alpha 2 and alpha 3 in a proportion of 84:12:4, respectively. Determination of ADP- and ouabain-binding site stoichiometry favours a theory with one substrate site per (alpha beta)2.
已采用离心法以高比活性测定猪肾中[14C]ADP和[3H]哇巴因与钠钾-ATP酶的结合。在钾离子存在的情况下，[14C]ADP结合数据对双位点模型的拟合给出了一个高亲和力组分，其占总位点的12±2%。该数值与50%有显著差异，即不能假定存在两个大小相等的组分。这与速率透析技术得到的1:1位点比例形成对比。这种差异可能是由于离心法能够在较低游离配体浓度下测定结合的ADP。如果在使用前不久对同位素进行纯化，在无钠离子情况下[3H]哇巴因的结合在Scatchard图中与一条直线相符。如果游离哇巴因的浓度不太高，哇巴因的非特异性结合可以忽略不计。在钠离子存在的情况下，等温线向上凹陷。对结合数据的分析给出了19:81%的划分，尽管尚未完全达到平衡。这是一个最大值，因为在游离哇巴因的小值时平衡的缺乏最为明显。因此，ADP结合研究得到了支持。此处的发现与半定量免疫测定结果在一定程度上一致，该测定表明猪肾酶分别含有比例为84:12:4的同工酶α1、α2和α3。ADP和哇巴因结合位点化学计量的测定支持了每个(αβ)2有一个底物位点的理论。
