CHARACTERISTICS OF ECTO-ATPASE OF XENOPUS OOCYTES AND THE INHIBITORY ACTIONS OF SURAMIN ON ATP BREAKDOWN

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (P-i) from the breakdown of extracellular ATP. Enzyme activity involved Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent dephosphorylation of ATP. Ca2+/Mg2+-dependent ecto-ATPase was active over a limited range of 0.01-1.0 mM ATP, while Ca2+/Mg2+-independent ATPase activity was active over a range of 0.1-30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0-9.0), but increased in a relatively linear manner with: (1) time of reaction (0-90 min), (2) number of cells (1-20 oocytes), and-(3) temperature (10-37 degrees C). Ecto-ATPase activity was unaffected by ouabain (100 mu M), sodium azide (100 mu M), and oligomycin (5 mu g/ml) (as inhibitors of endo-ATPases) and beta-glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methyl-thioATP>ADP, 2-methylthioADP, AMP>>alpha,beta-methylene ATP, beta,gamma-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P-2-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido)napthalene-1,3,5-trisulphonic acid), 100 mu M] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca2+/Mg2+-dependent enzyme. This striking property of suramin may point to a structural similarity between the ATP-binding sites of ecto-ATPase and purinoceptors, a potentially complicating factor where purinoceptors expressed in oocytes are used to test the potency of agonists and the efficacy of receptor antagonists and enzyme inhibitors.