Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart.
Sarcolemmal vesicles isolated from relaxed rat cardiac ventricles were 120-fold enriched in (Na+ + K+)-ATPase and 5'-nucleotidase activities (final recoveries, 50%). The alpha and beta chains of the former enzyme were visualized by the immunological approach. Inside-out sarcolemmal vesicles were isolated by affinity chromatography on immobilized concanavalin A. The yield of membranes was 0.45 mg of protein/g of muscle. The orientation of the unbound vesicles was studied by the increased accessibility of sarcolemma outer face markers (ouabain- and K+-binding sites, 5'-nucleotidase, and sialic acids) with permeability-increasing treatments: freeze-thaw cycles, sodium dodecyl sulfate, methanol, and valinomycin. The total ATP hydrolysis remained constant with a conversion of ouabain-insensitive activity into an ouabain-sensitive one. These agents caused a parallel increase in the ouabain sensitivity, the number of [3H]ouabain-binding sites, the monovalent cation stimulation of ATPase, and the 5'-nucleotidase activity. Valinomycin revealed that most vesicles were sealed to sequestered and exogenous K+. Inside-out vesicles were 80% pure in sidedness and sealing. The affinity chromatography did not affect the (Na+ + K+)-ATPase activity (200 mumol of product/mg of protein/h). This model of sarcolemma vesicles offers a new tool for ion transport studies.
D015720:5'-Nucleotidase; D000818:Animals; D002463:Cell Membrane Permeability; D002846:Chromatography, Affinity; D003208:Concanavalin A; D008297:Male; D009206:Myocardium / Q000648:ultrastructure*; D009708:Nucleotidases / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012508:Sarcolemma / Q000201:enzymology / Q000648:ultrastructure*; D012794:Sialic Acids / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D012794:Sialic Acids; D003208:Concanavalin A; D010042:Ouabain; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium