Adamo H P, Rega A F, Filoteo A G, Verma A K, Penniston J T
IQUIFIB-Facultad de Farmacia y Bioquímica (UBA), Cap. Fed, Buenos Aires, Argentina.
Biochim Biophys Acta. 1997 Feb 21;1324(1):85-90. doi: 10.1016/s0005-2736(96)00211-8.
A cDNA encoding a plasma membrane Ca2+ pump mutant V674P(ct120) was constructed and expressed in COS-1 cells. Immunoblots of transfected COS-1 membranes showed that the V674P(ct120) and the wild-type hPMCA4b(ct120) proteins were expressed at similar levels. The change of Val674 to Pro reduced the activity of the hPMCA4b(ct120) to an extent similar to that observed previously in the full-length Ca2+ pump (Adamo et al. (1995) J. Biol. Chem. 270, 30111-30114). Despite its lower activity, the apparent affinity for Ca2+ of the V674P(ct120) enzyme was at least as high as that of hPMCA4b(ct120), indicating that substitution of Val674 by Pro did not impair the interaction of the enzyme with Ca2+. The sensitivity of the V674P(ct120) enzyme to inhibition by vanadate was not significantly different from that of the hPMCA4b(ct120), supporting the idea that the mutation did not alter the equilibrium between E2-E1. The study of the Mg2+ dependency of the Ca2+ transport showed that the V674P(ct120) mutant reached maximum activation at 100 microM Mg2+ in contrast with 500 microM in the hPMCA4b(ct120). Furthermore, while at 2 mM Mg2+ the hPMCA4b(ct120) showed no sign of inhibition, the activity of the mutant decreased to less than 50% of the maximum activity observed at 100 microM Mg2+. These results indicate that the decrease in the activity observed upon substitution of Val674 by Pro was due to a higher sensitivity to Mg2+ as inhibitor.
构建了编码质膜Ca2+泵突变体V674P(ct120)的cDNA，并在COS-1细胞中进行表达。转染的COS-1细胞膜的免疫印迹显示，V674P(ct120)和野生型hPMCA4b(ct120)蛋白的表达水平相似。Val674突变为Pro使hPMCA4b(ct120)的活性降低，降低程度与之前在全长Ca2+泵中观察到的相似(Adamo等人(1995年)《生物化学杂志》270, 30111 - 30114)。尽管V674P(ct120)酶活性较低，但其对Ca2+的表观亲和力至少与hPMCA4b(ct120)一样高，这表明Pro取代Val674并不损害酶与Ca2+的相互作用。V674P(ct120)酶对钒酸盐抑制的敏感性与hPMCA4b(ct120)没有显著差异，支持了该突变未改变E2 - E1之间平衡的观点。对Ca2+转运的Mg2+依赖性研究表明，V674P(ct120)突变体在100μM Mg2+时达到最大激活，而hPMCA4b(ct120)在500μM Mg2+时达到最大激活。此外，在2 mM Mg2+时，hPMCA4b(ct120)没有抑制迹象，而突变体的活性降至在100μM Mg2+时观察到的最大活性的50%以下。这些结果表明，Val674被Pro取代后观察到的活性降低是由于对作为抑制剂的Mg2+更敏感。
