title: T-tubule membranes from chicken skeletal muscle possess an enzymic cascade for degradation of extracellular ATP.
abstract: The chicken T-tubule Mg2+-ATPase is an integral membrane glycoprotein that presents properties different from those of other ATPases located in skeletal muscle cells and exhibits ATP-hydrolysing activity on the extracellular side of the transverse tubule (TT) membranes. In this study we demonstrate that TT vesicles purified from chicken skeletal muscle possess ecto-ADPase and ecto-5'-nucleotidase activities that, along with ecto-ATPase, are able to sequentially degrade extracellular ATP to ADP, AMP and adenosine. Characterization studies of these TT ectonucleotidases revealed remarkable differences between ecto-ATPase and ecto-ADPase activities with respect to thermal stability, temperature dependence of the hydrolytic activity, effect of ionic strength, kinetic behaviour, divalent cation preference and responses to azide, N-ethylmaleimide, NaSCN, Triton X-100 and concanavalin A. Ecto-ATPase, but not ecto-ADPase, was inhibited by a polyclonal antibody against the chicken TT ecto-ATPase. On the basis of these results we propose that ATP and ADP hydrolysis are accomplished by two distinct enzymes and therefore the TT ecto-ATPase is not an apyrase. 5'-Nucleotidase activity was inhibited by adenosine 5'-[alpha,beta-methylene]diphosphate and concanavalin A, followed simple Michaelis-Menten kinetics and was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that AMP hydrolysis in T-tubules is catalysed by a typical ecto-5'-nucleotidase. Results obtained from electrophoresis experiments under native conditions suggest that ecto-ATPase, ecto-ADPase and 5'-nucleotidase might be associated, forming functional complexes in the T-tubule membranes. The TT ectonucleotidases constitute an enzymic cascade for the degradation of extracellular ATP that might be involved in the regulation of purinergic signalling in the muscle fibre.
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University, 28040 Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Delgado
authors.forename: G
authors.initials: G
authors.lastname: Moro
authors.forename: A
authors.initials: A
authors.lastname: Saborido
authors.forename: A
authors.initials: A
authors.lastname: Megías
mesh_terms: D015720:5'-Nucleotidase / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015703:Antigens, CD; D001081:Apyrase / Q000737:chemistry / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005110:Extracellular Space / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000648:ultrastructure; D009994:Osmolar Concentration; D013379:Substrate Specificity
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D002413:Cations, Divalent; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen