In vitro analysis of the general properties and junctional receptor characteristics of skeletal muscle membranes. Isolation, purification, and partial characterization of sarcolemmal fragments.
Muscle membranes were partially purified from rat leg muscles. Externally oriented membrane functions were used to monitor and characterize the resulting membrane fractions. Na(+)K(+)-stimulated Mg(++)-adenosinetriphosphatase, acetylcholinesterase, and cholinergic receptor activities are present and enriched in the density-gradient subfractions of crude sarcolemma when compared with the first pellet. The physical separation of the cholinesterase and receptor activities on the gradient subfractions is demonstrated. Receptor activity, determined by specific (125)I-labeled alpha-bungarotoxin binding, appears in fractions with densities similar to other plasma membranes (D(4) (20) 1.1015-1.1520). Acetylcholinesterase, on the other hand, is preferentially distributed in lighter density fractions (D(4) (20) 1.0507-1.0780) and parallels the gradient distribution of the ATPase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a high-molecular-weight glycoprotein sediments with the higher density fractions only. The data suggest a molecular dissection of the layers of the sarcolemma. The receptor is tentatively felt to be an integral component of the junctional plasma membrane. Acetylcholinesterase is felt to be superficially located on the ectolamina of the junctional sarcolemma, and may be woven within the matrix of the intersynaptic basement membrane.
D000110:Acetylcholinesterase / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001485:Basement Membrane; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D007457:Iodine Radioisotopes; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis* / Q000166:cytology; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011950:Receptors, Cholinergic; D012508:Sarcolemma / Q000032:analysis* / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate; D013570:Synaptic Membranes
D002038:Bungarotoxins; D006023:Glycoproteins; D007457:Iodine Radioisotopes; D011950:Receptors, Cholinergic; D012967:Sodium Dodecyl Sulfate; D012964:Sodium; D000110:Acetylcholinesterase; D000251:Adenosine Triphosphatases; D011188:Potassium