Gerbi A, Debray M, Maixent J M, Chanez C, Bourre J M
INSERM U. 26, Hôpital Fernand Widal, Paris, France.
J Neurochem. 1993 Jan;60(1):246-52. doi: 10.1111/j.1471-4159.1993.tb05844.x.
The Na+ sensitivity of whole brain membrane Na+,K(+)-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (alpha 1, low affinity for ouabain; alpha 2, high affinity; and alpha 3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10(-5) and 10(-7) M), we were able to discriminate Na+ sensitivity of Na+,K(+)-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, alpha 1, exhibited high Na+ sensitivity [Ka of 3.88 +/- 0.25 mM Na+ and a Hill coefficient (n) of 1.98 +/- 0.13]; the ouabain high-affinity isoform, alpha 2, had two Na+ sensitivities, a high (Ka of 4.98 +/- 0.2 mM Na+ and n of 1.34 +/- 0.10) and a low (Ka of 28 +/- 0.5 mM Na+ and an n of 1.92 +/- 0.18) Na+ sensitivity activated above a threshold (22 +/- 0.3 mM Na+); and the ouabain very-high-affinity isoform, alpha 3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.
利用哇巴因（α1，对哇巴因亲和力低；α2，对哇巴因亲和力高；α3，对哇巴因亲和力极高）的差异抑制作用，研究了全脑膜Na⁺，K⁺-ATP酶同工酶的Na⁺敏感性。在100 mM Na⁺浓度下，我们发现对哇巴因亲和力低、高和极高的同工型比例分别为21%、38%和41%。使用两种哇巴因浓度（10⁻⁵和10⁻⁷ M），我们能够通过非线性回归区分Na⁺，K⁺-ATP酶同工酶的Na⁺敏感性。哇巴因低亲和力同工型α1表现出高Na⁺敏感性[Na⁺的解离常数（Ka）为3.88±0.25 mM，希尔系数（n）为1.98±0.13]；哇巴因高亲和力同工型α2具有两种Na⁺敏感性，一种高敏感性（Na⁺的Ka为4.98±0.2 mM，n为1.34±0.10）和一种低敏感性（Na⁺的Ka为28±0.5 mM，n为1.92±0.18），在阈值（22±0.3 mM Na⁺）以上被激活；哇巴因极高亲和力同工型α3通过两个过程解析，似乎具有两种Na⁺敏感性（Na⁺的表观Ka值为3.5和20 mM）。我们表明，在没有哇巴因的情况下，Na⁺依赖性是至少五种Na⁺反应性的结果。膜中同工酶的这种分子功能特性可以解释归因于同工酶的生理作用的多样性。
