Isolation and characterization of the Mg 2+-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 Mg 2-ATPase and Ca 2+,Mg 2+-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 Mg 2+-ATPase activity was eluted with 0.43 M NaCl. The Ca 2+,Mg 2+-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 K m for MgATP in the range of 0.2 mM and a V m of 2.9 μmol P l • 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 32P i or [γ- 32P]ATP at significant levels when compared with the Ca 2+,Mg 2+-ATPase in an EGTA-containing medium. The kinetic pattern of the Mg 2+-ATPase was distinctly different from that of the Ca 2+,Mg 2+-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 Mg 2+-ATPase activity was detected. The Mg 2+-ATPase migrated much slower than the Ca 2+,Mg 2+-ATPase under non-denaturing conditions, whereas both enzymes had a molecular mass of 105 kDa on SDS gel electrophoresis.