title: A kinetic study of an unstable enzyme measured through coupling reactions. Application to the self-inactivation of detergent-solubilized Ca(2+)-ATPase from sarcoplasmic reticulum.
abstract: A methodology for the kinetic study of the self-inactivation of an unstable enzyme has been developed by using the transient-phase approach when the enzymatic activity is measured through a coupled enzyme system. An experimental design has been developed and applied to the inactivation of the Ca(2+)-ATPase from sarcoplasmic reticulum solubilized in the monomeric state. The catalytic activity of the ATP hydrolysis is determined in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes, and the oxidation of the last substrate, NADH, is continuously monitored. The experimental results show that both substrates, ATP and calcium, protect against enzyme inactivation. This enzyme, the monomeric ATPase, fulfills the catalytic cycle of the native ATPase, and free enzyme and first-calcium bound enzyme are proposed as the intermediates which are being inactivated.
authors.affiliation: Departamento de Bioquímica y Biología Molecular A, Edificio de Veterinaria, Universidad de Murcia, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Corbalán-García
authors.forename: J A
authors.initials: JA
authors.lastname: Teruel
authors.forename: R
authors.initials: R
authors.lastname: Varón
authors.forename: F
authors.initials: F
authors.lastname: García-Cánovas
authors.forename: J C
authors.initials: JC
authors.lastname: Gómez-Fernández
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003902:Detergents; D004789:Enzyme Activation; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D003902:Detergents; D009243:NAD; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D000252:Calcium-Transporting ATPases