Inoue A, Tonomura Y
J Biochem. 1982 Apr;91(4):1231-9. doi: 10.1093/oxfordjournals.jbchem.a133807.
Previously, several workers reported that at very low ionic strength and in the presence of ATP, the extent of binding of S-1 with the F-actin-tropomyosin-troponin complex or regulated actin (FA-TM-TN) is unaffected by removal of Ca2+. However, in this study we found that during the ATPase reaction at physiological ionic strength, the extent of binding of HMM with FA-TM-TN decreased markedly upon removal of CA2+. Therefore, the effects of Ca2+ were studied on the intermediate steps in the acto-HMM or acto-S-1 ATPase reaction. 1. The nucleotide-induced dissociation of acto-s-1 was studied using AMPPNP as substrate. The extent of binding of S-1 with regulated actin in the presence of Mg2+-AMPPNP increased in a sigmoidal manner as the S-1 concentration increased. When the molar ratio of actin monomer to S-1 was higher than 5-10, the removal of Ca2+ shifted the equilibrium of the dissociation reaction, FA-S-1-AMPPNP in equilibrium FA + S-1-AMPPNP, to the right. 2. The recombination rate of HMMPADP or S-1PADP with regulated actin in the absence of free Mg2+-ATP was estimated by measuring the time course of recovery in light-scattering intensity after addition of ATP. The rate decreased upon removal of Ca2+, when the molar ratio of actin monomer to S-1 was higher than 5-10. 3. The decomposition rate of HMMPADP was measured in the presence of Mg2+-ATP. In the absence of Ca2+, regulated actin did not affect this rate, whereas in its presence, regulated actin markedly accelerated the rate. These findings clearly indicated that at physiological ionic strength, removal of Ca2+ affects various elementary steps in the ATPase reaction to promote the dissociation of myosin heads from FA-TM-TN.
此前，有几位研究人员报告称，在极低离子强度且存在ATP的情况下，S-1与F-肌动蛋白-原肌球蛋白-肌钙蛋白复合物或调节性肌动蛋白（FA-TM-TN）的结合程度不受Ca2+去除的影响。然而，在本研究中我们发现，在生理离子强度下的ATP酶反应过程中，去除Ca2+后，重酶解肌球蛋白（HMM）与FA-TM-TN的结合程度显著降低。因此，研究了Ca2+对肌动蛋白-HMM或肌动蛋白-S-1 ATP酶反应中间步骤的影响。1. 以腺苷-5'-（β，γ-亚甲基）三磷酸（AMPPNP）为底物，研究了核苷酸诱导的肌动蛋白-S-1解离。随着S-1浓度的增加，在Mg2+-AMPPNP存在下，S-1与调节性肌动蛋白的结合程度呈S形增加。当肌动蛋白单体与S-1的摩尔比高于5-10时，去除Ca2+会使解离反应FA-S-1-AMPPNP⇌FA + S-1-AMPPNP的平衡向右移动。2. 通过测量添加ATP后光散射强度恢复的时间进程，估算了在无游离Mg2+-ATP情况下，HMMPADP或S-1PADP与调节性肌动蛋白的重组速率。当肌动蛋白单体与S-1的摩尔比高于5-10时，去除Ca2+后该速率降低。3. 在Mg2+-ATP存在下测量了HMMPADP的分解速率。在无Ca2+时，调节性肌动蛋白不影响该速率，而在有Ca2+时，调节性肌动蛋白显著加速了该速率。这些发现清楚地表明，在生理离子强度下，去除Ca2+会影响ATP酶反应中的各种基本步骤，从而促进肌球蛋白头部从FA-TM-TN上解离。
