Wright M V, van Houten J L
Department of Zoology, University of Vermont, Burlington 05405.
Biochim Biophys Acta. 1990 Nov 16;1029(2):241-51. doi: 10.1016/0005-2736(90)90160-p.
In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.
在草履虫中，尚未鉴定出具有钙泵特性的Ca2(+)-ATP酶。在此，我们报告了一种与表膜相关的Ca2(+)-ATP酶活性以及一种泵所特有的相应磷蛋白中间体。Ca2(+)-ATP酶活性需要3 mM镁以实现最佳的钙刺激（KCa = 90 nM），并且对ATP作为底物具有特异性（Km = 75 microM）。钒酸盐和平静霉素分别以约2 microM和0.5 microM的EC50抑制钙刺激的活性。同样，10 microM三氟拉嗪抑制80%的Ca2(+)-ATP酶活性，但牛钙调蛋白无法刺激该活性。Ca2(+)-ATP酶不受叠氮化钠（10 mM）、寡霉素（10微克/毫升）或哇巴因（0.2 mM）的抑制。用[γ-32P]ATP孵育表膜会以钙依赖、对羟胺敏感的方式特异性标记一种133 kDa的蛋白质，并且100 microM La3+会增加磷酸化水平。内质网富集部分的磷酸化标记一种与表膜蛋白不同的钙依赖蛋白，其分子量较低且不受La3+影响。表膜膜复合体的组成部分肺泡囊对钙的摄取与钙刺激的ATP水解耦合较差（钙转运/ATP水解小于0.2），并且与总Ca2(+)-ATP酶活性相比，对钒酸盐抑制的敏感性要低得多（EC50约为20 microM）。因此，大部分Ca2(+)-ATP酶活性可能与质膜相关。
