Isolation and characterization of the Mg2(+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations.
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.
Valente
A P
AP
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brasil.
Barrabin
H
H
Jorge
R V
RV
Paes
M C
MC
Scofano
H M
HM
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
D003902:Detergents; D013463:Sulfuric Acid Esters; D011092:Polyethylene Glycols; D017830:Octoxynol; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; C007482:dimethyl sulfate