Anderson S A, Jiang S, Mukkada A J
Department of Biological Sciences, University of Cincinnati, OH 45221.
Biochim Biophys Acta. 1994 Oct 12;1195(1):81-8. doi: 10.1016/0005-2736(94)90012-4.
The phosphorylated intermediate of a plasma membrane P-type ATPase in Leishmania donovani has been further characterized. The formation of the phosphorylated intermediate is sensitive to several ATPase inhibitors including vanadate, dicyclohexyl carbodiimide (DCCD), N-ethylmaleimide (NEM), and fluorescein isothiocyanate (FITC). These inhibitors affect purified immunoprecipitated protein as well as total plasma membrane fractions. Oligomycin, an inhibitor of mitochondrial ATPases, and ouabain, an inhibitor of Na+/K(+)-ATPases, had no effect on the formation of the phosphorylated intermediate. The ATPase phosphoprotein was acid stable and dephosphorylated at alkaline pH, indicating the presence of the acyl phosphate chemical linkage. Analysis of the phosphorylated amino acid by reduction with sodium boro[3H]hydride identified the residue as aspartate, confirming the formation of a beta-aspartyl phosphate intermediate. These data indicate the presence of a 105 kDa P-type ATPase on L. donovani plasma membrane that is mechanistically similar to to other P-type enzymes of higher eukaryotes.
杜氏利什曼原虫质膜P型ATP酶的磷酸化中间体已得到进一步鉴定。磷酸化中间体的形成对几种ATP酶抑制剂敏感，包括钒酸盐、二环己基碳二亚胺（DCCD）、N-乙基马来酰亚胺（NEM）和异硫氰酸荧光素（FITC）。这些抑制剂对纯化的免疫沉淀蛋白以及总质膜组分均有影响。线粒体ATP酶抑制剂寡霉素和Na+/K(+)-ATP酶抑制剂哇巴因对磷酸化中间体的形成没有影响。ATP酶磷蛋白在酸性条件下稳定，在碱性pH值下脱磷酸化，表明存在酰基磷酸化学键。用硼氢化钠[3H]还原分析磷酸化氨基酸，确定该残基为天冬氨酸，证实形成了β-天冬氨酰磷酸中间体。这些数据表明，杜氏利什曼原虫质膜上存在一种105 kDa的P型ATP酶，其作用机制与高等真核生物的其他P型酶相似。
