Benaim G, Romero P J
Centro de Biologia Celular, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela.
Biochim Biophys Acta. 1990 Aug 10;1027(1):79-84. doi: 10.1016/0005-2736(90)90051-o.
A subcellular fraction highly enriched in plasma membrane vesicles was prepared from Leishmania promastigotes. This fraction showed (Ca2+ + Mg2+)-ATPase activity. This, however, represented a small fraction (about 25%) of the overall ATPase activity. The Ca2(+)-ATPase showed general characteristics common to plasma membrane ATPases involved in Ca2+ transport. Thus, the Ca2(+)-ATPase was activated by Ca2+ with a high affinity (Km about 0.7 microM), saturating at about 5 microM Ca2+. Furthermore, it was stimulated by calmodulin (about 70-80% with 5 micrograms/ml) and almost fully inhibited by trifluoperazine (100 microM). The above vesicles accumulated Ca2+ against a concentration gradient and released it after the addition of A23187, as shown independently by 45Ca2+ and Arsenazo III studies. The transport mechanism showed the same kinetics parameters as described for the enzyme, indicating a single molecular entity. In addition, Ca2(+)-ATPase activity and Ca2+ uptake were completely inhibited by vanadate (20 microM), indicating that an E1-E2 type mechanism is involved. The results clearly demonstrate the presence of a Ca2+ pump in the plasma membrane of Leishmania which is capable of maintaining a low cytoplasmic Ca2+ concentration.
从利什曼原虫前鞭毛体中制备了一种高度富集质膜囊泡的亚细胞组分。该组分显示出（Ca2+ + Mg2+）-ATP酶活性。然而，这仅占总ATP酶活性的一小部分（约25%）。Ca2(+)-ATP酶表现出参与Ca2+转运的质膜ATP酶共有的一般特征。因此，Ca2(+)-ATP酶被Ca2+以高亲和力（Km约为0.7 microM）激活，在约5 microM Ca2+时达到饱和。此外，它受到钙调蛋白的刺激（5微克/毫升时约为70 - 80%），并几乎被三氟拉嗪（100 microM）完全抑制。如45Ca2+和偶氮胂III研究独立显示的那样，上述囊泡逆浓度梯度积累Ca2+，并在添加A23187后释放它。转运机制显示出与该酶描述的相同动力学参数，表明是单一分子实体。此外，钒酸盐（20 microM）完全抑制Ca2(+)-ATP酶活性和Ca2+摄取，表明涉及E1 - E2型机制。结果清楚地证明利什曼原虫质膜中存在一种Ca2+泵，它能够维持低细胞质Ca2+浓度。
