Nassi P, Marchetti E, Nediani C, Liguri G, Ramponi G
Dipartimento di Scienze Biochimiche, Università di Firenze, Italy.
Biochim Biophys Acta. 1993 Apr 8;1147(1):19-26. doi: 10.1016/0005-2736(93)90311-m.
Human red cell acylphosphatase actively hydrolyzes the Na+/K(+)-ATPase phosphoenzyme from erythrocyte membrane. This effect occurred with amounts of acylphosphatase (up to 10 units/mg membrane protein) within the physiological range, and the low value of the apparent Km (0.147 +/- 0.050 microM) indicates that the enzyme has a high affinity for this substrate. When added at the above concentration to inside out vesicles from human erythrocytes, acylphosphatase significantly enhanced the rate of strophantidine-sensitive ATP hydrolysis. The same amounts of acylphosphatase stimulated, although to a lower extent, the rate of ATP-dependent 22Na+ influx (normal efflux). Thus, the calculated stoichiometry for Na+/ATP was 2.68 in the absence of acylphosphatase and 1.06 in the presence of 10 units/mg vesicle protein of the enzyme. Conversely, acylphosphatase addition strongly decreased the rate of ATP-dependent 86Rb+(K+) efflux (normal influx) which, with 10 units/mg vesicle protein, was almost suppressed. As a consequence, the Na+/Rb+ ratio, calculated as 1.52 in the absence of acylphosphatase rose to 72.5 in the presence of 10 units/mg vesicle protein of this enzyme. These results suggest that, because of its hydrolytic activity on the phosphoenzyme intermediate, acylphosphatase 'uncouples' erythrocyte membrane Na+,K+ pump. Possible mechanisms for this effect are discussed.
人红细胞酰基磷酸酶可有效水解红细胞膜上的钠钾ATP酶磷酸化酶。在生理范围内，酰基磷酸酶的量（高达10单位/毫克膜蛋白）即可产生这种作用，其表观米氏常数（Km）较低（0.147±0.050微摩尔），表明该酶对该底物具有高亲和力。当以上述浓度添加到人红细胞内翻外小泡时，酰基磷酸酶显著提高了毒毛旋花子苷敏感的ATP水解速率。相同量的酰基磷酸酶虽然刺激程度较低，但也提高了ATP依赖的22Na+内流（正常外流）速率。因此，在不存在酰基磷酸酶时计算出的钠/ATP化学计量比为2.68，而在存在10单位/毫克小泡蛋白的该酶时为1.06。相反，添加酰基磷酸酶强烈降低了ATP依赖的86Rb+（K+）外流（正常内流）速率，当存在10单位/毫克小泡蛋白时，该速率几乎被抑制。结果，在不存在酰基磷酸酶时计算出的钠/铷比值为1.52，而在存在10单位/毫克小泡蛋白的该酶时升至72.5。这些结果表明，由于其对磷酸化酶中间体的水解活性，酰基磷酸酶使红细胞膜钠钾泵“解偶联”。文中讨论了这种作用的可能机制。
