Plasma Membranes from Muscle Tissue

Schapira, G; Dobocz, I.; Piau, Jean-Pierre; Delain, Etienne

doi:10.1016/b978-0-08-017822-6.50024-1

Cited by 2 publications

(2 citation statements)

References 18 publications

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“…This method results in a rather continuous distribution of material along the gradient and does not allow adequate separation of individual bands for our purposes. Virtually all other isolation procedures reported require either salt extraction (1,2) or incubation at elevated temperature (3) for successful purification of membranes.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation and characterization of muscle membranes using surface-specific labels.

Barchi¹,

Bonilla²,

Wong³

1977

Proc. Natl. Acad. Sci. U.S.A.

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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....

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Section: Resultsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%