McLaughlin J
Mol Biochem Parasitol. 1985 May;15(2):189-201. doi: 10.1016/0166-6851(85)90119-7.
Addition of Ca2+ (0.01-1 mM) to a standard Trypanosoma rhodesiense Mg2+-ATPase assay failed to elicit any increase in activity. However, in the absence of externally added Mg2+ and using calcium-EGTA or calcium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific Ca2+-ATPase. Cell fractionation studies revealed this ATPase to be predominantly associated with subcellular particles having an equilibrium density of 1.22 g cm-3 and identified as surface membrane. Using a discontinuous sucrose gradient, a surface membrane enriched (SME) fraction, only slightly contaminated with mitochondria as judged by dichlorophenolindophenol-linked alpha-glycerophosphate dehydrogenase activity, was prepared. The SME fraction exhibited Ca2+-ATPase activity, using 200 nM free Ca2+, of 90 and 21 mU mg-1 protein, respectively, using CDTA and EGTA as buffering ligands. This latter result was most unexpected and indicated that the Ca2+-ATPase, in addition to having no Mg2+ requirement, was inhibited by submicromolar levels of Mg2+. The Ca2+-ATPase was found to have a K0.5 = 128 +/- 22 nM free Ca2+, the response to increasing Ca2+ concentration displaying an extremely high degree of co-operativity (Hill number (nH) = 4.9). The enzyme was found to be highly substrate-specific for ATP with K0.5 = 6.2 +/- 0.61 microM ATP. A Hill plot of the reaction velocity as a function of ATP concentration indicated two substrate binding sites (nH = 1.55). A range of potential modulators of ATPase activity were investigated, with only vanadate (V2O3-8) having any effect: 47% inhibition at 5.0 microM. The Ca2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (50 microM), whilst addition of calmodulin failed to produce any stimulation of activity. It is concluded that the kinetic properties of this ATPase are compatible with a potential role in the regulation of intracellular Ca2+ in bloodstream T. rhodesiense.
在标准的罗得西亚锥虫镁离子 - ATP酶检测中添加钙离子（0.01 - 1 mM）未能引起活性增加。然而，在不添加外源镁离子的情况下，使用钙 - 乙二醇双四乙酸（EGTA）或钙 - 环己二胺四乙酸（CDTA）精确维持游离金属离子浓度时，可以测量到一种特异性的钙离子 - ATP酶。细胞分级分离研究表明，这种ATP酶主要与平衡密度为1.22 g/cm³的亚细胞颗粒相关，这些颗粒被鉴定为表面膜。使用不连续蔗糖梯度，制备了一个表面膜富集（SME）组分，通过二氯酚靛酚偶联的α - 甘油磷酸脱氢酶活性判断，该组分仅轻微受线粒体污染。使用CDTA和EGTA作为缓冲配体时，SME组分在200 nM游离钙离子存在下分别表现出90和21 mU/mg蛋白质的钙离子 - ATP酶活性。后一个结果非常出乎意料，表明该钙离子 - ATP酶除了不需要镁离子外，还受到亚微摩尔水平镁离子的抑制。发现该钙离子 - ATP酶的K0.5 = 128 ± 22 nM游离钙离子，对钙离子浓度增加的反应显示出极高的协同性（希尔系数（nH）= 4.9）。发现该酶对ATP具有高度底物特异性，K0.5 = 6.2 ± 0.61 μM ATP。反应速度作为ATP浓度函数的希尔图表明有两个底物结合位点（nH = 1.55）。研究了一系列可能的ATP酶活性调节剂，只有钒酸盐（V2O3 - 8）有任何作用：在5.0 μM时抑制47%。该钙离子 - ATP酶不受钙调蛋白拮抗剂氯丙嗪（50 μM）和三氟拉嗪（50 μM）的影响，而添加钙调蛋白未能产生任何活性刺激。得出的结论是，这种ATP酶的动力学特性与在罗得西亚锥虫血流形式中调节细胞内钙离子的潜在作用相符。
