Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation.
Transverse tubule vesicles were isolated from frog skeletal muscle by a procedure initially described by Rosemblatt et al. (J. Biol. Chem. 256, 8140-8148 (1981)) and later modified by Hidalgo et al. (J. Biol. Chem. 258, 13937-13945 (1983]. A large fraction of the isolated vesicles (80-90%) were sealed, as indicated by the detergent induced increase in (Na+ + K+)-ATPase activity and ATP-dependent ouabain binding. To determine the orientation of the sealed vesicles binding of digoxin, a lipid soluble derivative of ouabain, was measured. The same values of ATP-dependent digoxin binding were found with or without detergents, indicating that all the vesicles that are sealed have the ATP site accessible, and hence are sealed with the cytoplasmic side-out (inside-out orientation). The transverse tubule preparation isolated from frog muscle is highly purified, as indicated by its cholesterol content and its (Na+ + K+)-ATPase activity; negligible contamination with sarcoplasmic reticulum was observed, as indicated by the protein composition and the lack of measurable Ca2+-ATPase activity of the isolated transverse tubules. High initial rates of Mg2+-ATPase activity were found, with the peculiar property of being inhibited during the course of the reaction. Addition of lysophosphatidylcholine or saponin partially prevented the inhibition of Mg2+-ATPase activity during the reaction.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002784:Cholesterol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D008563:Membrane Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D009132:Muscles / Q000648:ultrastructure*; D010042:Ouabain / Q000494:pharmacology; D011898:Ranidae; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D008563:Membrane Lipids; D008565:Membrane Proteins; D010042:Ouabain; D002784:Cholesterol; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase