Alterations in the natural abundance 13C NMR spectra of skeletal muscle membranes depending on the extraction medium of muscle

Minced rat muscles were extracted with NaCl solution with additions: 1) 10 mM EDTA, or 2) 2 mM MgCl2, or 3) None (final ionic strength equivalent to 150 mM NaCl, pH 7.0). The total muscle membranes were pelleted between 1500 and 252,000 x g. All membranes were washed several times by homogenization with 0.05 M NaCl, pH 6.1, and centrifugation at 252,000 x g. The final pellets were suspended in 2H2O to contain 150 mg membrane protein/ml in 30% 2H2O. 13C NMR spectra were recorded at 50.3 MHz, under fully relaxed conditions. The spectral pattern varied according to the extraction medium of muscle: Relative to None addition, EDTA caused a decrease in the intensity of the polyunsaturated carbon resonance (128.6 ppm), whereas with MgCl2 the opposite effect was found. Metal analysis, after combustion of the membranes, showed 1.1-1.6 mol Ca2+ per 105 g protein in None and Mgcl2 membranes, and 1000-times less Ca2+ in EDTA membranes. The Mg2+ content of the membranes was not affected by EDTA, however, it was increased 5-fold when MgCl2 was present in the extraction medium. Thus, the results indicate that membrane-bound Ca2+ and Mg2+ alter the conformation of membrane-phospholipis