Pfliigers Arch - Eur J Physiol (1995) 429:412-418 9 Springer Verlag 1995 Airat U. Ziganshin 9 Lilia E. Ziganshina..Brian E King Geoffrey Bttrnstock Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown Received: 19 May 1994/Received after revision: 11 July 1994/Accepted: 19 July 1994 Abstract Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phos- phate (Pi) from the breakdown of extracellular ATE En- zyme activity involved Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent dephosphorylation of ATE Ca2+/Mg2+-dependent ecto-ATPase was active over a limited range of 0.01-1.0 mM ATP, while Ca2+/Mg2+-in - dependent ATPase activity was active over a range of 0.1-30 mM ATE 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) tem- perature (10--37~ Ecto-ATPase activity was unaffected by ouabain (100gM), sodium azide (100gM), and oligomycin (5 gg/ml) (as inhibitors of endo-ATPases) and fl-glycerophosphate (10mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alka- line phosphatase). Total ecto-ATPase activity was re- duced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential sub- strates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methyl- thioATP>ADP, 2-methylthioADP, AMP>>o~,fl-methylene ATP, fl,?'-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-methylbenz- amido)napthalene-l,3,5-trisulphonic acid), 100 gM] sig- nificantly inhibited total ecto-ATPase activity; this inhi- bition was competitive for the Ca2+/Mg2+-dependent en- zyme. This striking property of suramin may point to a structural similarity between the ATP-binding sites of ecto-ATPase and pufinoceptors, a potentially complicat- A. U. Ziganshin - L. E. Ziganshina 9 B. E King G. Burnstock (~) Depat~nent of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT, UK A. U. Ziganshin 9 L. E. Ziganshina Kazan Medical Institute, 49 Butlerov Street, Kazan, 420012, Russia ing factor where purinoceptors expressed in oocytes are used to test the potency of agonists and the efficacy of receptor antagonists and enzyme inhibitors. Key words Xenopus laevis 9 Oocytes Ecto-ATPase 9 Purine nucleotides Pyrimidine nucleotides 9 Suramin Introduction Xenopus laevis oocytes represent a robust and efficient surrogate system for the expression of foreign proteins when messenger RNA (mRNA) is injected into oocyte cytoplasm and translated [19]. These amphibian cells have been used extensively in the functional study of proteins that insert into the plasmalemma and act as either extracellular receptors, or ion channels, or both (for reviews, see [38, 40]). Recently, Xenopus oocytes have been used to study the pharmacological properties of receptor subtypes for extracellular ATP (P2-purinoceptors). Four of five major subtypes of Pz-purinoceptors (P2u, Pzx, P2y and Pzz) have been cloned, and expression studies carried out using oo- cytes. These receptors include: a Pzu-purinoceptor, from a murine neuroblastoma x rat glioma hybrid (NG108-15) cell line [14, 26]; a Pzx-purinoceptor, from guinea-pig vas deferens [36]; a Pzy-purinoceptor, from guinea-pig brain [21]; a P2ypurinoceptor, from chick brain [4, 43]; a P2z-purinoceptor, from routine macrophages [33]; and unclassified P2-purinoceptors, from a human macro- phage (HL60) leukaemic cell line [31] and a murine macrophage-like (J774) cell line [20]. A Pz-purinoceptor resembling the P2t-subtype has also been cloned [4]. Whether cloned or studied in situ, the classification of all major P2-purinoceptor subtypes has been based large- ly on the relative potencies of purine and pyrimidine nu- cleotides [1, 9]. Apart from a paucity of selective antago- nists for P2-purinoceptors, the classification of ATP re- ceptors has been hindered further by the presence of ex- tracellular enzymes (ecto-ATPases) that degrade ATP