title: Isolation and characterization of muscle membranes using surface-specific labels.
abstract: Membranes were purified from rat muscle by a differential centrifugation method that avoids the use of salt extraction or incubations at elevated temperature. Three populations of membrane-limited vesicles were defined having average densities of 1.112 (fraction I), 1.141 (fraction II), and 1.158 (fraction III) g/ml in a continuous sucrose gradient. Lactoperoxidase-catalyzed iodination of intact muscle prior to isolation of membranes resulted in highest specific activity in fraction I, although all fractions could be equally labeled after isolation. 125I-Labeled wheat germ agglutinin incubated at low concentration with intact muscle preferentially labeled fraction I. Parallel studies on previously isolated fractions indicated that fraction I also contained the highest concentration of potential receptors for wheat germ agglutinin. In experiments on whole muscle, concanavalin A bound predominantly to sarcolemma with slight variable binding to T-tubular and nuclear membrane but no binding to sarcoplasmic reticulum or mitochondria. Parallel binding studies with isolated membrane fragments indicated heavy binding of concanavalin A by membranes in fraction I with scattered binding in fractions II and III. Na+K+Mg2+ ATPase was specifically enriched in fraction I but was also present in fraction II in a proportion similar to 125I labeling. Ca2+ ATPase was most active in fraction II but present in significant levels in fraction I. It is concluded from these and other data that fraction I contains predominantly sarcolemma membrane, while T-tubular membrane may represent a significant component of fraction II. Ca2+ ATPase activity in fraction I is intrinsic to the sarcolemma.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D003208:Concanavalin A / Q000378:metabolism; D007784:Lactoperoxidase; D037102:Lectins; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D012508:Sarcolemma / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D000345:Affinity Labels; D037102:Lectins; D003208:Concanavalin A; D007784:Lactoperoxidase; D000251:Adenosine Triphosphatases