Brooker R J, Slayman C W
J Biol Chem. 1983 Jul 25;258(14):8827-32.
We have shown previously (Brooker, R.J., and Slayman, C.W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226) that the plasma membrane [H+]-ATPase of Neurospora crassa is inhibited by N-ethylmaleimide (NEM), which reacts at an essential nucleotide-protectable site on the Mr = 104,000 polypeptide. The present study demonstrates that Mg2+ has a biphasic effect on NEM inhibition. At low concentrations (0.01-0.1 mM, Mg2+ decreases the sensitivity of the enzyme to NEM, while at high concentrations (greater than 1 mM), it enhances sensitivity. These effects are seen in the presence or absence of nucleotides (ATP, ADP). Mg2+ also acts in a concentration-dependent way to influence the degradation of the ATPase by trypsin. Low concentrations of Mg2+ have little or no effect on tryptic inactivation of ATPase activity or on the disappearance of the Mr = 104,000 polypeptide and the stepwise appearance of Mr = 100,000 and 91,000 tryptic fragments. High concentrations of Mg2+ decrease the rate of inactivation, and a new fragment of Mr = 98,000 is seen. Taken together, the NEM and trypsin results indicate that the Neurospora [H+]-ATPase possesses high and low affinity Mg2+ binding sites which affect the conformation of the enzyme. The divalent cation specificity of the sites has also been investigated. Co2+, Mn2+, and (to a lesser extent) Ni2+ mimic the behavior of Mg2+, but Ca2+ has a different effect, at least at the high affinity site. It appears to bind to that site, based on its ability to inhibit ATP hydrolysis (in the presence of Mg2+), but does not offer protection against NEM inhibition. The results suggest a way in which Ca2+ may serve as a physiological regulator of the ATPase.
我们之前已经表明（布鲁克，R.J.，和斯莱曼，C.W.（1982年）《生物化学杂志》257卷，12051 - 12055页；布鲁克，R.J.，和斯莱曼，C.W.（1983年）《生物化学杂志》258卷，222 - 226页），粗糙脉孢菌的质膜[H⁺]-ATP酶受到N - 乙基马来酰亚胺（NEM）的抑制，NEM在分子量为104,000的多肽上一个必需的核苷酸可保护位点发生反应。本研究表明Mg²⁺对NEM抑制具有双相效应。在低浓度（0.01 - 0.1 mM）时，Mg²⁺降低了酶对NEM的敏感性，而在高浓度（大于1 mM）时，它增强了敏感性。在有或没有核苷酸（ATP、ADP）的情况下都能观察到这些效应。Mg²⁺还以浓度依赖的方式影响胰蛋白酶对ATP酶的降解。低浓度的Mg²⁺对ATP酶活性的胰蛋白酶失活或分子量为104,000的多肽的消失以及分子量为100,000和91,000的胰蛋白酶片段的逐步出现几乎没有影响。高浓度的Mg²⁺降低了失活速率，并出现了一个分子量为98,000的新片段。综合来看，NEM和胰蛋白酶的结果表明粗糙脉孢菌[H⁺]-ATP酶具有高亲和力和低亲和力的Mg²⁺结合位点，这些位点影响酶的构象。还研究了这些位点的二价阳离子特异性。Co²⁺、Mn²⁺以及（在较小程度上）Ni²⁺模仿了Mg²⁺的行为，但Ca²⁺至少在高亲和力位点有不同的作用。基于其抑制ATP水解的能力（在有Mg²⁺存在时），它似乎能结合到那个位点，但不能提供针对NEM抑制的保护。结果提示了Ca²⁺可能作为ATP酶生理调节剂的一种方式。
