Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown

Abstract

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°C). Ecto-ATPase activity was unaffected by ouabain (100 μM), sodium azide (100 μM), and oligomycin (5 μg/ml) (as inhibitors of endo-ATPases) and β-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-methylthioATP>ADP, 2-methylthioADP, AMP≫α,β-methylene ATP, β,γ-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 P₂-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido) napthalene-1,3,5-trisulphonic acid), 100 μM] 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.