Improved techniques for separating muscle cell membranes from solubilized muscle proteins
A new process solubilizes muscle proteins making it possible to separate membranes with their oxidizable phospholipids. Isolated cod muscle membranes sedimented at 4,000 × g for 15 min at pH 5 or below but not at pH 6 or higher. Isolated membranes added to the supernatant of homogenized muscle solubilized at pH 3 and centrifuged at 10,000 × g for 30 min also sedimented at 4,000 × g for 15 min. This was in contrast to the membranes naturally present in solubilized homogenate. This suggests that factors other than viscosity were involved in sedimentation.
Most membrane phospholipid in cod or herring muscle homogenates solubilized at pH 3 or 10.5 could not be sedimented by centrifugation at 4,000 or 10,000 × g for 15 min. Addition of two high Mw chitosans (50–190 and 310–375 kDa) increased membrane removal while two low Mw chitosans (1 and 33 kDa) did not. The high Mw chitosans had to be added prior to solubilization of the muscle homogenates to be effective.
Calcium chloride, and to a lesser extent MgCl2, aided in membrane removal from muscle homogenates solubilized at pH 3 in the presence of citric acid or malic acid but not lactic acid. Adding the citric acid and Ca 2+ before solubilizing the muscle homogenates was needed for the effect. At 1 mM citric acid, 70–80% of the phospholipid and 25–30% of the protein were removed at 10 mM Ca2+. At 8 mM Ca2+ , citric acid showed an optimal effect on phospholipid removal at 5 mM with 90% of the phospholipid and 35% of the protein removed from homogenates solubilized at pH 3. The percentage of phospholipid and protein removed from muscle homogenates solubilized at pH 10.5 increased with increasing Ca 2+ concentrations at 1 mM citric acid. At 8 mM Ca2+, addition of citric acid at 5 mM improved membrane removal from muscle homogenates solubilized at pH 10.5 to about 80% from 58% in its absence. Ca2+ and citric acid might exert their influence by disconnecting linkages between membranes and cytoskeletal proteins and/or aiding in aggregation of the membranes.