A high affinity Ca 2+-dependent ATPase in the surface membrane of the bloodstream stage of Trypanosoma rhodesiense

Abstract

Addition of Ca 2+ (0.01–1 mM) to a standard Trypanosoma rhodesiense Mg 2+-ATPase assay failed to elicit any increase in activity. However, in the absence of externally added Mg 2+ and using calcium-EGTA or calcium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific Ca 2+-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 α-glycerophosphate dehydrogenase activity, was prepared. The SME fraction exhibited Ca 2+-ATPase activity, using 200 nM free Ca 2+, 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 Ca 2+-ATPase, in addition to having no Mg 2+ requirement, was inhibited by submicromolar levels of Mg 2+. The Ca 2+-ATPase was found to have a K 0.5 − 128 ± 22 nM free Ca 2+, the response to increasing Ca 2+ concentration displaying an extremely high degree of co-operativity (Hill number ( n H) = 4.9). The enzyme was found to be highly substrate-specific for ATP with K 0.5 = 6.2 ± 0.6 μM ATP. A Hill plot of the reaction velocity as a function of ATP concentration indicated two substrate binding sites ( n H = 1.55). A range of potential modulators of ATPase activity were investigated, with only vanadate (V 2O 8 3−) having any effect: 47% inhibition at 5.0 μM. The Ca 2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine (50 μM) and trifluoperazine (50 μM), 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 Ca 2+ in bloodstream T. rhodesiense.