Allen B G, Katz S, Roufogalis B D
Division of Pharmacology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Biochem J. 1987 Jun 15;244(3):617-23. doi: 10.1042/bj2440617.
Formation of the phosphorylated intermediate (ECaP) of the human erythrocyte Ca2+-stimulated ATPase (Ca2+-ATPase) was more rapid and reached steady state sooner at 400 microM-Ca2+ than at 1 microM-Ca2+. Calmodulin increased the apparent rate of ECaP formation at 1 microM-Ca2+, whereas at 400 microM-Ca2+, calmodulin decreased the steady-state level of the ECaP without affecting its apparent rate of formation. Removal of endogenous Mg2+ with trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid, which decreased both the velocity and Ca2+-sensitivity of the Ca2+-ATPase, did not alter the Ca2+-sensitivity or the apparent rate of formation of ECaP. ECaP formation at high Ca2+ concentrations was not affected by Mg2+ concentrations as high as 1 mM, and the ECaP could be dephosphorylated by ADP and ATP along either the forward or reverse pathways. The results suggest that high Ca2+ concentrations inhibit Ca2+-ATPase activity by preventing dephosphorylation of the E2P complex, rather than by inhibition of the transformation from E1CaP ('high-Ca2+-affinity' ECaP) to E2CaP ('lower-energy' ECaP).
人红细胞钙刺激ATP酶(Ca2+-ATP酶)磷酸化中间体(ECaP)的形成在400微摩尔/升钙离子浓度下比在1微摩尔/升钙离子浓度下更快,且更快达到稳态。钙调蛋白在1微摩尔/升钙离子浓度下增加了ECaP形成的表观速率,而在400微摩尔/升钙离子浓度下,钙调蛋白降低了ECaP的稳态水平,但不影响其表观形成速率。用反式-1,2-二氨基环己烷-N,N,N',N'-四乙酸去除内源性镁离子,这降低了Ca2+-ATP酶的速度和钙敏感性,但没有改变ECaP的钙敏感性或表观形成速率。在高钙离子浓度下ECaP的形成不受高达1毫摩尔/升镁离子浓度的影响,并且ECaP可以通过ADP和ATP沿着正向或反向途径去磷酸化。结果表明,高钙离子浓度通过阻止E2P复合物的去磷酸化来抑制Ca2+-ATP酶活性,而不是通过抑制从E1CaP(“高钙亲和力”ECaP)到E2CaP(“低能量”ECaP)的转变。