title: The beta-aspartyl phosphate intermediate in a Leishmania donovani promastigote plasma membrane P-type ATPase.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Cincinnati, OH 45221.
authors.forename: S A
authors.initials: SA
authors.lastname: Anderson
authors.forename: S
authors.initials: S
authors.lastname: Jiang
authors.forename: A J
authors.initials: AJ
authors.lastname: Mukkada
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines; D007893:Leishmania donovani / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D014638:Vanadates / Q000494:pharmacology
chemical_list: D006898:Hydroxylamines; D010750:Phosphoproteins; C032197:beta-aspartyl phosphate; D019811:Hydroxylamine; D001224:Aspartic Acid; D014638:Vanadates; D000251:Adenosine Triphosphatases; D016650:Fluorescein-5-isothiocyanate; D005033:Ethylmaleimide