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 couldbe equally labeled after isolation. l25I-Labeled wheat germ aegutinin 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 predominautly 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. Progress toward an understanding of the molecular aspects of excitable membrane function has been restricted by the difficulty of preparing pure fractions of this membrane from readily available tissues. Muscle represents a potential source of homogeneous excitable membrane, but its exploitation for this purpose has been limited by problems encountered in isolation, including the large amount of connective tissue that it contains, the presence of high concentrations of insoluble contractile protein, and the presence of other extensive membrane systems' including the sarcoplasmic reticulum and T-tubular network.Most attempts to isolate purified muscle surface membrane have relied on prolonged salt extraction in LiBr or KBr to solubilize the contractile protein elements (1, 2). Others have used incubation at elevated temperatures to effect this separation (3). For experiments in which the composition and vectorial organization of proteins within the membrane are to be studied, theoretical reservations to prolonged salt extraction techniques can be raised, including the potential removal of extrinsic membrane proteins usually associated with the sarcolemma and possible reorientation of intrinsic proteins within the membrane. Action of endogenous proteases on membrane proteins during incubation at 30-40' would also be undesirable in such experiments....

Na+-K+Pump Regulation and Skeletal Muscle Contractility

“…In the sarcolemma of rabbit skeletal muscle, Mitchell et al (299) obtained a Na ϩ -K ϩ -ATPase activity of 57 m ⅐ h Ϫ1 ⅐ mg protein Ϫ1 , rather close to that of t-tubular membranes (39 mol ⅐ h Ϫ1 ⅐ mg protein Ϫ1 ). In rat skeletal muscle, the Na ϩ -K ϩ -ATPase activity in a membrane fraction containing primarily t tubules was 58% of that in the fraction containing sarcolemma (20). Thus, as already pointed out by Hidalgo et al (206), the contents of Na ϩ -K ϩ -ATPase in t tubules and sarcolemma are not as different as suggested by the [ 3 H]ouabain binding studies by Venosa and Horowicz (422).…”

Characteristics of saxitoxin binding to the sodium channel of sarcolemma isolated from rat skeletal muscle.

“…This binding site density is comparable to that obtained by others in rat diaphragm and in frog skeletal muscle. I8Otaed 8arcolemma In membrane preparations from rat skeletal muscle, the highest specific activities of sarcolemmal markers are found in a light membrane fraction banding between 22 and 25 % sucrose following equilibrium centrifugation on a continuous density gradient (Barchi et al 1977(Barchi et al , 1979. The highest level of specific [3H]STX binding was also uniformly distributed through this fraction (Barchi et al 1979).…”

“…Work in muscle, however, has been limited to studies employing whole muscle or crude homogenate and has been hampered by the low level of binding obtained and by problems of interpretation in regard to surface geometry (Almers & Levinson, 1975;Ritchie & Rogart, 1977b;Jaimovich, Venosa, Shrager & Horowitz, 1976). Methods for the preparation of isolated muscle surface membrane have been refined to the point where binding studies on purified sarcolemma can be undertaken (Barchi, Wong & Bonilla, 1977; Barchi, Weigele, Chalikian & Murphy, 1979).…”

“…Membrane was prepared from the mixed fibre type and fast muscles of the rat hind leg. Purity of isolated 384 Na CHANNEL IN SARCOLEMMA 385 membranes was monitored using marker enzyme assays and cholesterol determinations as previously reported (Barchi et al 1977(Barchi et al , 1979. Purified sarcolemma was suspended in Tris buffer (5 mM, pH 7.3) and stored frozen at -20 'C until needed.…”

Tri-partite complex for axonal transport drug delivery achieves pharmacological effect

“…In part, this reflects the fact that WGA adheres to muscle cell membranes [36] as well as being taken up by adsorptive endocytosis at nerve termini. This also explains a depot effect whereby additional amounts of the compound continue to be introduced into the axonal stream over a period of several days.…”