Hsieh W L, Pierce W S, Sze H
Department of Botany, University of Maryland, College Park, Maryland 20742.
Plant Physiol. 1991 Dec;97(4):1535-44. doi: 10.1104/pp.97.4.1535.
Ca(2+)-ATPases keep cytoplasmic [Ca(2+)] low by pumping Ca(2+) into intracellular compartments or out of the cell. The transport properties of Ca(2+)-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca(2+) transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase) partially inhibited oxalate-stimulated Ca(2+) transport activity; however, it had no effect on calmodulin-stimulated Ca(2+) uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca(2+)-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma(32)P]ATP resulted in the formation of a single acyl [(32)P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca(2+), but independent of Mg(2+). Its enhancement by La(3+) is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca(2+) transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca(2+) transport, similar to their effect on the erythrocyte plasma membrane Ca(2+)-ATPase. These results would indicate that the calmodulin-stimulated Ca(2+) transport originated in large part from a plasma membrane-type Ca(2+) pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca(2+)-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested.
Ca(2+)-ATP酶通过将Ca(2+)泵入细胞内区室或细胞外，从而使细胞质中的[Ca(2+)]保持在低水平。对胡萝卜（胡萝卜品种丹弗斯）组织培养细胞中Ca(2+)泵ATP酶的转运特性进行了研究。与内质网标记物共迁移的囊泡中依赖ATP的Ca(2+)转运受到钙调蛋白的刺激，增加了三到四倍。环匹阿尼酸（肌浆网/内质网Ca(2+)-ATP酶的特异性抑制剂）部分抑制了草酸盐刺激的Ca(2+)转运活性；然而，它对由ATP或GTP驱动的钙调蛋白刺激的Ca(2+)摄取没有影响。结果表明存在两种类型的Ca(2+)-ATP酶，即内质网型和质膜型。有趣的是，用[γ(32)P]ATP孵育膜会导致形成一种120千道尔顿的单一酰基[(32)P]磷蛋白。这种磷蛋白的形成依赖于Ca(2+)，但不依赖于Mg(2+)。La(3+)对其的增强作用是质膜型Ca-ATP酶磷酸化酶中间体的特征。W-7（一种钙调蛋白拮抗剂）、ML-7（肌球蛋白轻链激酶抑制剂）或甲状腺素会降低钙调蛋白刺激的Ca(2+)转运。酸性磷脂，如磷脂酰丝氨酸，刺激Ca(2+)转运，类似于它们对红细胞质膜Ca(2+)-ATP酶的作用。这些结果表明，钙调蛋白刺激的Ca(2+)转运很大程度上源自一种120千道尔顿的质膜型Ca(2+)泵。有人提出，在内膜（如内质网和分泌囊泡）以及质膜上可能存在钙调蛋白刺激的Ca(2+)-ATP酶。
