Sarcolemmal membranes from hamster and chicken skeletal muscle: Isolation and chemical composition

• 1.1. Plasma membranes were obtained from hamster (Mesocricetus auratus Wateth.) and chicken (Callus gallus L.). Skeletal muscle was isolated by muscle homogenization, protein extraction by inorganic salt solutions (0.4 M LiBr and 0.6 M KCl) and differential centrifugation. After purification on a discontinuous sucrose gradient, several fractions were obtained. The upper fraction (20% sucrose, w/w) yielded in the form of vesicles by electron microscope examination.
• 2.2. (Na⁺ + K⁺ )-ATPase and 5'-nucleotidase as plasma membrane markers were found to be concentrated in the upper fraction. Practically no succinate dehydrogenase activity was detected.
• 3.3. By means of polyacrylamide gel electrophoresis it was possible to separate 7–8 protein bands the molecular weights of which range from 26,000 to 200,000 daltons; and 4 bands for glycoproteins.
• 4.4. The ratio lipid: protein and the molar ratio cholesterol :phospholipid were found to be 0.93–0.94 and 0.25–0.38, respectively.
• 5.5. Glucose, mannose, galactose and fucose, hexosamines and sialic acids were determined in these preparations of membranes.

     

Isolation of sarcolemmal membranes from cardiac muscle

• 1.1. Sarcolemmal membranes from cardiac tissue were isolated to a high degree of purity by brief extraction of homogenate with KC1 followed by 2 successive discontinuous sucrose density gradients of extracted particles.
• 2.2. Sarcolemmal membranes contained 40% adenylate cyclase, 22% guanylate cyclase and 20% ouabain-sensitive (Na⁺−K⁺) ATPase: contamination of sarcolemma by other intracellular membranes was negligible.
• 3.3. Electron microscopic examination of membranes showed presence of relatively empty vesicles of various shapes and sizes while electrophoretic analysis revealed about 20 protein bands of which 6 were prominent.

     

Isolation and characterization of plasma membranes from rabbit skeletal muscle

An improved procedure was developed for the isolation of skeletal muscle plasma membranes. This method includes a DNAse treatment of the homogenate prior to the isolation of membranes by differential and sucrose gradient centrifugation techniques. We obtained two light fractions which were highly enriched in many biochemical and chemical plasma membrane markers. These fractions were shown to be mostly inside-out vesicles containing a Ca2+-ATPase activity. These results suggested that this enzyme could participate in the extrusion of calcium ions from the muscle cells.     

Isolation and composition of thick filaments from rabbit skeletal muscle

A method has been developed for the isolation of thick filaments from rabbit skeletal muscle. We found that the thick filaments of this muscle are readily dispersed in the presence of a relaxing medium if the M and Z-line structures are first extracted in a low-salt solvent system. Thick filaments were separated from thin filaments by zone sedimentation in a 10% to 30% glycerol density gradient. The isolated filaments are homogeneous in length (1.5 to 1.6 μm) and retain the physical characteristics of these structures observed in sectioned muscle. Gel electrophoresis of thick filaments in the presence of sodium dodecyl sulfate showed a band of C-protein as well as bands with mobilities characteristic of the heavy and light chains of myosin. No other protein species was detected in these experiments. Thus our results provide evidence against the presence of a special protein component which would serve as the core of the skeletal thick filament structure. From the relative stain density of bands, the molar ratio of C-protein to myosin was estimated to be 1 to 5.8.     

Lipid composition of purified transverse tubule membranes isolated from amphibian skeletal muscle

The level and proportion of lipids and their fatty acid composition were analyzed in highly purified transverse tubule membranes of amphibian skeletal muscle. Tubule membranes show (a) a higher content of lipids, (b) a higher phospholipid/cholesterol ratio and (c) a different phospholipid composition from other subcellular fractions, such as the light and heavy membranes from sarcoplasmic reticulum, which are similar in lipid profile. Transverse tubule membranes are characterized by a high percentage of phosphatidylserine and sphingomyelin and a low proportion of phosphatidylcholine compared with the other membranes. All three show a high proportion of ethanolamine plasmalogens (50% of the total ethanolamine glycerophospholipid). Transverse tubule membrane lipids contain a high proportion of 20- and 22-carbon polyunsaturated fatty acids, predominantly 20:4, 20:5, 22:5 and 22:6. Arachidonate predominates in phosphatidylinositol, eicosapentaenoate and docosahexaenoate in ethanolamine and serine glycerophospholipids.     

Isolation of skeletal muscle plasmatic membranes as closed vesicles

Ultrasonic treatment of skeletal muscle sarcolemma preparations in 0.25 M sucrose, 0.1 M NaCl, 0.001 M tris-HCl-buffer (pH 7.4) induces formation of closed osmotically active vesicles which may be isolated by centrifugation of a sonicated suspension within the sucrose density gradient. The vesicles retain the enzymic activity peculiar to the initial sarcolemma preparations.     

Lactate transport by skeletal muscle sarcolemmal vesicles

Recent studies have indicated that lactate traversal of the sarcolemmal membrane of skeletal muscle could be a carrier mediated process. In the present study, the initial rates of L(+)-lactate flux (J_lact) were measured in highly purified rat hindlimb skeletal muscle sarcolemmal vesicles. Fluxes were determined by the vesicle uptake of L(+)-[U-¹⁴C] lactate from the extra-vesicular medium. J_lact was saturable with respect to increasing concentrations of L(+)-lactate. Regression of these data to the Michaelis-Menten equation yielded a Km of 12.5 mM. J_lact was inhibited 81% by 10 mM pyruvate and 83% by 5mM alpha-cyano 4 hydroxycinnamate (p<0.05), but not by D-lactate indicating the presence of a stereoselective monocarboxylate transporter in the sarcolemmal membrane. Preincubation of the vesicles with the protein modifier, N-ethylmaleimide (20mM), inhibited J_lact by 86% (p<0.05). An inhibitor of the inorganic anion exchanger, SITS (1mM), had no effect on J_lact. However, J_lact was markedly sensitive to an inwardly directed proton gradient (p<0.05), and the flux was more closely related to the concentration of external ionic L(+)-lactate than to the protonated (HLa) form. These studies suggest that skeletal muscle sarcolemmal membranes possess a specific transport system for L-lactate and other monocarboxylates, which has similar properties to the lactate carrier described for several other tissues.     

Phosphorylase phosphatase from skeletal muscle membranes

Microsomes containing 12-15 U/mg phosphorylase phosphatase were obtained from skeletal muscle glycogen particles following glycogen digestion and differential centrifugation. The phosphatase associated with the membranes is in an inhibited state; dilution induces dissociation and deinhibition of the enzyme. Phosphatase-depleted membranes can rebind purified phosphatase catalytic subunit but not the complex between catalytic subunit and inhibitor 2. Binding involves a receptor, deduced from saturation phenomena, which is responsible for inhibition of the bound enzyme and which is a protein, since trypsin treatment releases all bound enzyme and prevents rebinding. The phosphatase extracted from the membranes is of type 1 and is a mixture of complexes, the major ones displaying a Mr of 300,000 and 70,000. From these complexes the 35-kDa catalytic subunit can be obtained either by trypsin treatment or by acetone precipitation. Purification to homogeneity involves chromatography on polylysine and FPLC chromatography on Mono Q and Polyanion SI columns. The purified enzyme exhibits a specific activity of 26,800 U/mg (27,900 U/mg after trypsin treatment) and consists of a major protein of 38 kDa (SDS gel electrophoresis). A minor component of 33 kDa, which may represent either a proteolytic product or an isozyme, can be separated. Both 38-kDa and 33-kDa catalytic subunits form a 70-kDa inactive complex with inhibitor 2 and upon incubation of the complexes the catalytic subunit is slowly converted to the inactive conformation which can then be reactivated by either the kinase FA or trypsin and Mn2+. Alternatively the inactive catalytic subunit is reactivated by Mn2+ alone once it has been isolated by FPLC chromatography on SI. The observation that the same catalytic subunit is present at various cell locations (namely cytosol, glycogen particles and microsomes), though in different conformations, is in favour of the hypothesis that displacement of the catalytic subunit from one cell site to the other may represent a new mechanism for phosphatase regulation in skeletal muscle.     

Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle

Two types of myosin isolated from ordinary (fast) and dark (slow) muscles of carp were examined by ATPase and in vitro motility assays. Vmax of the ATPase activity and sliding velocity of ordinary myosin showed 1.6 and 1.5 times higher activities than those of dark myosin, whereas those of mammalian fast myosin were much higher, 3 to 10 times, than those of slow myosin. Although ordinary myosin had almost identical activities to those of mammalian fast myosin, activities of dark myosin was twice of those of mammalian slow myosin. This high motile activity of dark myosin can account for the physiological role of dark muscle in cruising of fish. By comparing Km of the actin-activated ATPase activity, ordinary myosin was appeared to have higher affinity to F-actin than dark myosin, and this was confirmed by the binding assay of HMM or S-1 of carp myosin to F-actin. Investigation of myosin assembly by electron microscopy and the centrifugation assay revealed that ordinary myosin assembled much poorly than dark myosin or mammalian fast myosin. This phenomenon may reflect characteristic cellular function of fish skeletal muscle.     

Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor

When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.     

Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles

Summary The Ca²⁺ permeability of rabbit skeletal muscle sarcolemmal vesicles was investigated by means of radioisotope flux measurements. A membrane vesicle fraction highly enriched in sarcolemma, as revealed by enzymatic markers, was obtained from the 22–27% region of sucrose gradients after isopycnic centrifugation. The ability of sarcolemmal vesicles to exchange Na⁺ for Ca²⁺ was investigated by measuring Ca²⁺ influx into and efflux from sarcolemmal vesicles in the presence and absence of a Na⁺ gradient. It was found that Ca²⁺ movements were enhanced in the direction of the higher Na⁺ concentration. When intra- and extravesicular Na⁺ concentrations were high, Na⁺–Na⁺ exchange predominated and Na⁺–Ca²⁺ exchange was low or absent. The presence of the Ca²⁺ ionophore A23187 in the dilution medium resulted in the rapid release of Ca²⁺ and the elimination of the Na⁺-enhanced efflux of Ca²⁺, suggesting that internal rather than bound external Ca²⁺ was exchanged with Na⁺. La³⁺ abolished Na⁺–Ca²⁺ exchange and decreased overall membrane permeability. Na⁺–Ca²⁺ exchange was not due to sarcoplasmic reticulum or mitochondrial contaminants. This investigation suggests that skeletal muscle, like cardiac muscle and neurons, is capable of a transmembranous Na⁺–Ca²⁺ exchange.     

Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle.

Fast (extensor digitorum longus) and slow (soleus) rat skeletal muscles served as the source for isolation and biochemical comparison of two distinct surface membrane fractions with properties of the sarcolemma and transverse tubular system. Enriched sarcolemmal membrane from soleus demonstrated a lighter density after sucrose density centrifugation. Sialic acid content was 1.5-fold higher in soleus (62 nmol/mg) than extensor (40 nmol/mg). The specific activity of (Na+ + K+ + Mg2+)-ATPase was similar (1.40 and 1.65 micronmol Pi/mg per 5 min) with the soleus enzyme displaying a (1) greater resistance to inhibition by ouabain, and (2) broader ionic ratio (Na+/K+) requirement than extensor enzyme. The polypeptide and phospholipid composition showed no major differences between the two muscle types. The second surface membrane fraction, tentatively identified as transverse tubule, differed in membrane composition. The major polypeptide of extensor was of 95 000 molecular weight whereas for soleus a Mr=28 000 species was dominant. Total phospholipid content of soleus was 1.5-fold greater than extensor due mostly to increased levels of phosphatidylcholine and phosphatidylethanolamine. Endogenous membrane protein kinase for the 28 000 molecular weight polypeptide was found exclusively in this membrane. The reaction conditions were identical for extensor and soleus since both required divalent cations (Ca2+ and Mg2+) and neither was affected by cyclic AMP. Soleus showed a 2-fold higher capacity for phosphate incorporation than extensor. These studies show that surface membrane fractions derived from fast and slow muscles differ in terms of functional and compositional properties. These differences are specific not only for the surface membrane but for the muscle type and may relate to the known physiological differences observed between fast and slow mammalian muscle.