ISOLATION AND CHARACTERIZATION OF THE MG-2+-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 μmol Pl • 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 [γ-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. © 1990.