Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

Degradation of skeletal muscle plasma membrane proteins by calpain

“…For the present studies, this inhibitor was employed at a final concentration of 1 mM instead of the lower concentration used in earlier assays because the higher concentration permits the reagent to penetrate even "sealed" muscle membrane vesicles and to inhibit Na +, K+-ATPase regardless of the insideoutside orientation of the vesicles [30,45]. Preliminary tests at various concentrations of sucrose revealed that under the hypotonic conditions of the earlier assays [39] the membrane vesicles were permeable to 0.2 mM ouabain, but that under isotonic conditions the higher concentration of reagent was needed for full inhibition. Valinomycin, a K+-selective ionophore [30,45] was Resolution of calpains and calpastatin of frog muscle on DEAE-cellulose.…”

“…By electron microscopy, the frog muscle plasma membrane preparation appears to consist largely of small, closed, unilamellar vesicles [39]. The sidedness and leakiness of these vesicles was tested in the presence of 0.15 M sucrose, which, together with other salts and reagents, provided an isotonic medium (equivalent to 0.19 M sucrose) for frog tissues.…”

“…The sidedness and leakiness of these vesicles was tested in the presence of 0.15 M sucrose, which, together with other salts and reagents, provided an isotonic medium (equivalent to 0.19 M sucrose) for frog tissues. Na +, K+-ATPase was assayed by a sensitive coupled-enzyme procedure in which the ATPase activity is characteristically inhibited by ouabain [39]. For the present studies, this inhibitor was employed at a final concentration of 1 mM instead of the lower concentration used in earlier assays because the higher concentration permits the reagent to penetrate even "sealed" muscle membrane vesicles and to inhibit Na +, K+-ATPase regardless of the insideoutside orientation of the vesicles [30,45].…”

“…Female Rana pipiens, 7-9 cm long, obtained from St. Croix Biological Supply (Stillwater, MN), were maintained in pans of water at 5~ Plasma membrane vesicles were isolated from transverse tubules of frog skeletal muscle by methods that have been described [39] and suspensions were stored at -200C. Protein concentrations [25] in the three preparations ranged from 3.5 to 4.5 mg/ml.…”

“…Protein concentrations [25] in the three preparations ranged from 3.5 to 4.5 mg/ml. Transverse tubules were used as the source of plasma membranes because in frog skeletal muscle these delicate structures constitute the largest area of plasma membrane in the ceils and provide the most easily accessible source of isolated membranes that are free of basal lamina [37,39,40]. Suspensions of sealed erythrocyte ghosts (5.0 mg protein/ml) and of unsealed ghosts (3.4 mg protein/ml), which are more readily permeable to proteins, were made according to procedures of Steck [46] from human blood that had been stored for six weeks at a blood bank.…”