title: Isolation and characterization of the Mg2(+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations.
abstract: Preparations of sarcoplasmic reticulum vesicles, obtained according to the method of Eletr and Inesi (Biochim. Biophys. Acta (1972) 282, 174), contained both Mg2(+)-ATPase and Ca2+, Mg2(+)-ATPase activity. The two enzymes were solubilized by a mixture of digitonin and lysophosphatidylcholine and separated on a DEAE-cellulose column eluted with a discontinuous gradient of NaCl. The Mg2(+)-ATPase activity was eluted with 0.43 M NaCl. The Ca2+,Mg2(+)-ATPase was obtained by increasing the NaCl concentration of the elution medium to 0.40 M. The fraction eluted with 0.043 M NaCl was insensitive to micromolar concentrations of calcium, resistant to oligomycin, ouabain, orthovanadate and thiocyanate, and was inhibited by low concentrations of Triton X-100. The enzyme showed a single apparent Km for MgATP in the range of 0.2 mM and a Vm of 2.9 mumol Pi.min-1.mg-1 protein. Activity was maximal over a broad peak between pH 6.0-8.0. Hydrolysis of ATP was unaffected by dimethylsulfoxide concentrations up to 20% (v/v) and was inhibited at higher concentrations. The enzyme was not phosphorylated by either 32Pi or [gamma-32P]ATP at significant levels when compared with the Ca2+,Mg2(+)-ATPase in an EGTA-containing medium. The kinetic pattern of the Mg2(+)-ATPase was distinctly different from that of the Ca2+,Mg2(+)-ATPase under the same conditions. The fraction eluted from the DEAE-cellulose column was subjected to electrophoresis under non-denaturing conditions. Only one band with Mg2(+)-ATPase activity was detected. The Mg2(+)-ATPase migrated much slower than the Ca2+,Mg2(+)-ATPase under non-denaturing conditions, whereas both enzymes had a molecular mass of 105 kDa on SDS gel electrophoresis.
authors: Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brasil.
authors: A P
authors: AP
authors: Valente
authors: H
authors: H
authors: Barrabin
authors: R V
authors: RV
authors: Jorge
authors: M C
authors: MC
authors: Paes
authors: H M
authors: HM
authors: Scofano
mesh_terms: D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000302:isolation & purification / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013463:Sulfuric Acid Esters / Q000494:pharmacology
chemical_list: D003902:Detergents; D013463:Sulfuric Acid Esters; D011092:Polyethylene Glycols; D017830:Octoxynol; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; C007482:dimethyl sulfate