Identification of a new subpopulation of triad junctions isolated from skeletal muscle; Morphological correlations with intact muscle

Summary It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca²⁺-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, weak, and breakage resistant, strong, triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.     

Bridging structures spanning the junctioning gap at the triad of skeletal muscle

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.     

Purification of dystrophin from skeletal muscle

Dystrophin was purified from rabbit skeletal muscle by alkaline dissociation of dystrophin-glycoprotein complex which was first prepared by derivatized lectin chromatography. Dystrophin-glycoprotein complex was isolated from digitonin-solubilized rabbit skeletal muscle membranes by a novel two-step method involving succinylated wheat germ agglutinin (sWGA) chromatography and DEAE-cellulose ion exchange chromatography. Proteins co-purifying with dystrophin were a protein triplet of Mr 59,000 and four glycoproteins of Mr 156,000, 50,000, 43,000, and 35,000, all previously identified as components of the dystrophin-glycoprotein complex. Alkaline treatment of sWGA/DEAE-purified dystrophin-glycoprotein complex resulted in complete dissociation of the dystrophin-glycoprotein complex. In order to separate dystrophin from its associated proteins, alkaline-dissociated dystrophin-glycoprotein complex was sedimented by sucrose gradient centrifugation. The residual glycoproteins which contaminated peak dystrophin-containing gradient fractions were then removed by WGA-Sepharose adsorption. The resulting protein appeared as a single band with an apparent Mr of 400,000 on overloaded Coomassie Blue-stained gels. The absence of WGA-peroxidase staining on nitrocellulose transfers of the pure protein indicated that the pure protein was devoid of contaminating glycoproteins. Antisera raised against the carboxyl terminus of human skeletal muscle dystrophin (which does not cross-react with the carboxyl terminus of the chromosome 6-encoded dystrophin-related protein) recognized the pure protein as did antisera specific for the amino terminus of human dystrophin. These data indicate that the protein isolated is indeed the intact, predominant skeletal muscle isoform product of the Duchenne muscular dystrophy gene.     

Purification of desmin from adult mammalian skeletal muscle.

A method has been developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate-filament protein [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-urea yielded desmin that was routinely more than 97% 55 000-dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that the relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10nm filaments have not been unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.     

Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle

Two enzymes which possess 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities have been purified from pig skeletal muscle. One of the enzymes corresponds to type M phosphoglycerate mutase. The other enzyme shows properties similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase present in mammalian erythrocytes. The erythrocyte and the muscle enzyme possess the same molecular (56 000) and subunit (27 000) weights. The synthase, phosphatase and mutase activity ratio is similar in both enzymes, and they are affected by the same inhibitor (glycerate 3-P) and activators (glycolate 2-P, pyrophosphate, sulfite and bisulfite).     

AgNOR proteins from morphologically intact isolated nucleoli.

AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by "standard" methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.     

Purification and characterization of the parvalbumin from monkey skeletal muscle

1. A high affinity Ca2+ binding and low mol. wt protein, parvalbumin, was purified from monkey skeletal muscle. 2. As compared with other animals, only one component and a lower content of monkey parvalbumin were found. 3. This may suggest that both the component and the content of parvalbumin decreases with biological evolution. 4. The parvalbumin was found to have a mol. wt of 11,400, a pI of 5.1, a high aspartic acid and lysine content, maximum absorption at around 260 nm, a blocked amino-terminal, an immunological distinction, 2 mol Ca2+ binding/mol, and a conformational change by Ca2+ binding. 5. Parvalbumin was shown to have alpha type properties.     

Purification and characterization of adenosine deaminase from camel skeletal muscle

Adenosine deaminase was purified (780-fold) from skeletal muscle of camel (Camelus Dormedarius) to homogeneity level by using DEAE Sephadex chromatography, ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The enzyme appeared to be monomeric with subunit molecular weight of 43kDa and isoelectric point of 4.85. The enzyme showed specificity for adenosine and exhibited Michaelis-Menten Kinetics with kappa(cat) of 1112.41 min(-1) and K(m) of 14.7 microM at pH 7.5. The pH and temperature optima for enzyme activity were 7-7.5 and 25 degrees C, respectively. Free energy (DeltaG*), enthalpy (DeltaH*) and entropy (DeltaS*) of activation for denaturation of adenosine deaminase at 50 degrees C were 88.94, 99.65 kJmol(-1) and 33.16 Jmol(-1), respectively. The purified enzyme had half-lives of 636 and 61 min at 25 and 50 degrees C, respectively. The activation energy for catalysis of camel skeletal muscle adenosine deaminase was 9.13 kJmol(-1). Free energy (DeltaG#), enthalpy (DeltaH#) and entropy (DeltaS#) of activation for hydrolysis of adenosine deaminase at 25 degrees C were 50.35, 6.65 kJmol(-1) and -146.62 Jmol(-1), respectively. Purine riboside inhibited the enzyme competitively with K(i) of 16 microM.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Purification and characterization of cathepsin B from monkey skeletal muscle

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.     

Purification of a latent protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (Mr = 260K) has been isolated from rabbit skeletal muscle. The enzyme has a very low activity towards phosphorylase a isolated from the same tissue, but its activity towards this substrate is stimulated several fold after dissociation by 2-mercaptoethanol treatment. The purified phosphatase shows one major protein staining band on non denaturing polyacrylamide gel electrophoresis, and contains four subunits with molecular weights of 95K, 75K, 65K and 38K. The catalytic activity resides in the Mr = 38K subunit and is not sensitive to inhibition by the heat stable protein phosphatase inhibitor-1 or modulator protein. Polyamines stimulate the holoenzyme in a dose dependent, biphasic manner, but inhibit the activity of the dissociated Mr = 38K catalytic subunit.     

Purification and crystallization of creatine kinase from rabbit skeletal muscle

Crystallization is the primary rate-limiting step in protein structure determination. It has been our experience over approximately 10 years that crystals are obtained in about 20% of the proteins attempted and that only about 10% of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization of microheterogeneity in a protein regarded as pure by conventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of precipitants, crystallization conditions, and additives failed to discover conditions from which usable crystals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution.     

Purification and autophosphorylation of insulin receptors from rat skeletal muscle

Insulin receptors of rat skeletal muscle were purified by first extracting a plasma membrane-enriched pellet obtained from a muscle homogenate with Triton X-100, followed by WGA-Sepharose and insulin-Sepharose affinity chromatography. Routinely, 4-5 micrograms of purified receptor were obtained from 15 g of tissue. The purified receptors are composed of two major polypeptides with molecular weights of 130,000 and 95,000, respectively. The binding of [125I]insulin by the purified receptors was analyzed by a Scatchard plot. There are at least two binding components. The high-affinity component, with an apparent association constant (Ka) of 2.0 X 10(9) M-1, comprises 10% of the total insulin binding sites; while the low-affinity component, with a Ka value of 1.4 X 10(8) M-1, represents 90% of the binding sites. Assuming the insulin receptor to have a molecular weight of 300,000, the receptor binds 1.7 mol of insulin per mol at saturation. Insulin is capable of stimulating the autophosphorylation of the beta-subunit of the muscle insulin receptor (Mr 95,000) by 5-10-fold. The stoichiometry of this phosphorylation reaction was determined as 0.8 phosphate per insulin binding site after a 10 min incubation with 100 nM insulin. In a previous report, I showed that the insulin stimulation of glucose transport in diaphragms from neonatal rats was small, even although the diaphragms had normal levels of insulin receptors and glucose transporters (Wang, C. (1985). Proc. Natl. Acad. Sci. USA 82, 3621-3625). To determine whether or not receptor autophosphorylation might be related to this insensitivity to insulin, the level of receptor phosphorylation was quantitated in diaphragms from rats at different stages of development. Autophosphorylation remains unchanged from birth to 21 days of age, suggesting that the lower insulin-stimulated glucose uptake by diaphragms at early stages of postnatal development as compared to that by diaphragms of older rats, is not due to a difference in receptor kinase.     

Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle

Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500-2800 pmol/mg protein (60-70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin--Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps. The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.     

Purification of glycogen phosphorylase from small quantities of mouse skeletal muscle

A new approach to the purification of skeletal muscle glycogen phosphorylase is described. The purification scheme is particularly suited to preparation of the enzyme from small amounts of tissue. A combination of dye-ligand chromatography and hydrophobic chromatography yields homogenous enzyme with good recoveries. The purification is rapid and may be completed in a working day.