Separation of plasma membrane markers by glycerol-induced blistering of muscle cells

Abstract

Glycerol (50%, w/w) was found to cause blistering of chick primary myoblast and fibroblast plasma membranes and extensive blistering of 5--6-day-old-myotube plasma plasma membranes in tissue culture. The tips of myoblasts and fibroblasts appeared to be the most sensitive portion of the plasma membrane to the blistering effect of glycerol. The glycerol-induced blistering of myotubes was reduced and delayed by brief EDTA pretreatment. Glycerol treatment (50, 15 and 8% sequentially) of myotubes was used to remove plasma membrane blisters and a plasma membrane-enriched fraction was isolated from these blisters using a modified Dextran T500-polyethylene-glycol 6000 aqueous two-phase polymer system. This fraction was found to be enriched 4.1-fold for 5'-nucleotidase activity, but not for other putative plasma membrane markers, (Na+ + K+)-ATPase activity or alpha-[125I]bungarotoxin binding material. Autoradiographs of alpha-[125I]bungarotoxin, glycerol-treated (50%, w/w) myotubes showed the plasma membrane blisters to be devoid of reduced silver grains. 5'-Nucleotidase was shown to be an ectoenzyme on myoblasts and 5-day-old myotubes and the total cellular activity was present on the cell surface. During the period of myoblast fusion and myotube formation, cell surface activity decreased to a low level while total cellular activity was elevated.