Plasma membranes from cultured muscle cells: isolation procedure and separation of putative plasma-membrane marker enzymes.
Partially purified plasma membranes were obtained from chick-embryo muscle cells grown in tissue culture. The purification procedure involved homogenization in buffered isotonic sucrose followed by differential and sucrose density gradient centrifugations. The activities of five plasma-membrane markers, as well as microsomal and mitochondrial markers, were followed throughout the purification. When cultures were labeled with [(125)I]alpha-bungarotoxin, which binds to the surface of cultured muscle cells, the distributions of bound alpha-bungarotoxin and Na(+),K(+)-ATPase (EC 3.6.1.3) activity were nearly identical. The activities of these two plasma-membrane markers were maximal in the upper two fractions of the sucrose density gradient and were purified 5- to 7-fold with respect to total particulate protein. These fractions contained 20-30% of the Na(+),K(+)-ATPase activity and bound alpha-bungarotoxin, 4% of the microsomal marker TPNH-dependent cytochrome c reductase, 0.2% of the mitochondrial marker succinate-dependent cytochrome c reductase, 2.7% of the cellular RNA, and 0.02% of the DNA. The activity of the commonly used plasma-membrane marker, 5'-nucleotidase (EC 3.1.3.5), was low in the upper two sucrose gradient fractions and was maximal in a more dense fraction. The distributions of the other two plasma-membrane markers, leucyl beta-naphthylamidase and phosphodiesterase I, were intermediate between Na(+),K(+)-ATPase and 5'-nucleotidase. The distributions of all markers were similar in preparations from cultures containing mononucleated myogenic cells, multinucleated myotubes, fibroblasts, or all three cell types. Modification of the procedure to include homogenization in the absence of sucrose resulted in a 3.4-fold purification of the membranes containing 5'-nucleotidase, which were shifted to a lower density.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000626:Aminopeptidases / Q000302:isolation & purification; D000818:Animals; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002038:Bungarotoxins; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D003579:Cytochrome Reductases / Q000302:isolation & purification; D005467:Floxuridine / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008722:Methods; D008861:Microsomes / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology; D009132:Muscles / Q000166:cytology* / Q000201:enzymology; D009708:Nucleotidases / Q000302:isolation & purification*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification; D011485:Protein Binding; D013347:Subcellular Fractions / Q000201:enzymology
D002038:Bungarotoxins; D007457:Iodine Radioisotopes; D005467:Floxuridine; D003579:Cytochrome Reductases; D009708:Nucleotidases; D010727:Phosphoric Diester Hydrolases; D000626:Aminopeptidases; D000251:Adenosine Triphosphatases; D001973:Bromodeoxyuridine