Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.     

Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes

PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 microM in oocytes or 0.25 microM in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTPA activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase alpha inhibits the activated PTPase activity (I50 = 5 microM). The PTPase activity of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C. J., Parker, P. J., Hermann, J., Waterfield, M. D., & Merlevede, W. (1988) Biochem. J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., & Merlevede, W. (1989) Eur. J. Biochem. 180, 15-22].     

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.     

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.     

Isolation of a new high molecular weight protein associated with desmin and vimentin filaments from avian embryonic skeletal muscle

Filaments with a diameter of 80-120 A have been prepared from 14-d-old chick embryonic skeletal muscle, using a physiological salt solution and gel filtration chromatography. The filaments obtained are composed of the two known muscle intermediate-filament proteins, vimentin and desmin, as well as the vimentin- and desmin-associated high molecular weight protein, synemin (230,000 mol. wt). In addition, they contain a previously unidentified high molecular weight protein (280,000 mol wt) which differs from synemin by isoelectric point, molecular weight, and immunological reactivity. Immunofluorescence on cultured myogenic cells,using antisera to the 280,000-dalton polypeptide, has revealed that this protein has the same spatial distribution as desmin, vimentin, and synemin in both early myotubes, where it associates with cytoplasmic filaments, and late in myotubes, where it is associated with myofibril Z lines. Examination by immunofluorescence of frozen sections of developing embryonic skeletal muscle reveals a gradual diminution in the presence of the 280,000-dalton protein. The 280,000-dalton protein is undetectable in adult skeletal and smooth muscle, as shown by immunofluorescence and immunoautoradiography. In chick embryonic fibroblasts grown in tissue culture, only a subpopulation of the cells is reactive with antibodies to the 280,000-dalton protein even though all these cells contain vimentin. In the reactive cells, vimentin and the 280,000-dalton polypeptide exhibit an indistinguishable cytoplasmic filamentous network, which aggregates into filamentious bundles when the cells are exposed to colcemid. These results suggest that this newly identified high molecular weight protein is closely associated with intermediate filaments containing either vimentin alone or vimentin, desmin and synemin. The expression of this protein appears to be developmentally regulated and does not appear to parallel the expression of any of the other three intermediate-filament proteins. The absence of the 280,000-dalton polypeptide in adult muscle cells and its gradual reduction during development implies that is probably not required for the maintenance of Z-disk structure after the assembly of the sarcomere.     

Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.     

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.     

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.     

Isolation and characterization of rabbit skeletal muscle protein phosphatases C-I and C-II

Previous studies have shown that phosphorylase phosphatase can be isolated from rabbit liver and bovine heart as a form of Mr approximately 35,000 after an ethanol treatment of tissue extracts. This enzyme form was designated as protein phosphatase C. In the present study, reproducible methods for the isolation of two forms of protein phosphatase C from rabbit skeletal muscle to apparent homogeneity are described. Protein phosphatase C-I was obtained in yields of up to 20%, with specific activities toward phosphorylase a of 8,000-16,000 units/mg of protein. This enzyme represents the major phosphorylase phosphatase activity present in the ethanol-treated muscle extracts. The second enzyme, protein phosphatase C-II, had a much lower specific activity toward phosphorylase a (250-900 units/mg). Phosphatase C-I and phosphatase C-II had Mr = 32,000 and 33,500, respectively, as determined by sodium dodecyl sulfate disc gel electrophoresis. The two enzymes displayed distinct enzymatic properties. Phosphatase C-II was associated with a more active alkaline phosphatase activity toward p-nitrophenyl phosphate than was phosphatase C-I. Phosphatase C-II activities were activated by Mn2+, whereas phosphatase C-I was inhibited. Phosphatase C-I was inhibited by rabbit skeletal muscle inhibitor 2 while phosphatase C-II was not inhibited. Both enzymes dephosphorylated glycogen synthase and phosphorylase kinase, but displayed different specificities toward the alpha- and beta-subunit phosphates of phosphorylase kinase (Ganapathi, M. K., Silberman, S. R., Paris, H., and Lee, E. Y. C. (1980) J. Biol. Chem. 246, 3213-3217). The amino acid compositions of the two proteins were similar. Peptide mapping of the two proteins showed that they are distinct proteins and do not have a precursor-proteolytic product relationship.     

Separation and characterization of two phosphorylase phosphatase inhibitors from rabbit skeletal muscle.

Two heat-stable and trypsin-labile inhibitors of phosphorylase phosphatase, designated inhibitor-1 and inhibitor-2, were partially purified from extracts of rabbit skeletal muscle by heating and coloumn chromatography using DEAE-dellulose and Bio-gel P-60. Inhibitor-1 exists in an active phosphorylated form and an inactive dephosphorylated form. The interconversion of phosphorylated inhibitor-1 and dephosphorylated inhibitor-1 is mediated by protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and a Mn2+-stimulated phosphoprotein phosphatase. Inhibitory activity of inhibitor-2 is not influenced by treatment with either the kinase or the Mn2+-stimulated phosphatase. The molecular weights of inhibitor-1 and inhibitor-2 estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis are 26000 and 33000 respectively. Both inhibitor-1 and inhibitor-2 inhibit phosphorylase phosphatase by a mechanism which appears to be non-competitive with respect to the substrate phosphorylase a. Inhibitor fractions at early stages of purification also inhibit cyclic-AMP-dependent histone phosphorylation, but this kinase inhibitory activity resides with a protein moiety which is separable from inhibitor-1 and inhibitor-2.     

Isolation and characterization of two 70 kDa modulator-complexes from rabbit skeletal muscle

The activation as well as the inactivation of the ATP,Mg-dependent protein phosphatase has been shown to be totally dependent upon the presence of the modulator subunit. This modulator (inhibitor-2) is a heat stable protein and its isolation in pure form (32 kDa) always includes a boiling step. The boiled modulator fractions are known to be inhibitory to the phosphatase activity. Unboiled rabbit skeletal muscle preparations do not contain "free modulator", but two higher molecular weight complexes (70 kDa) can be isolated which have the 32 kDa modulator together with a 38 kDa protein. One complex is the already characterized inactive ATP,Mg-dependent phosphatase [FCM] while the second one, [MX], although seemingly of identical composition, does not exhibit phosphatase activity when measured under the usual conditions. The MX-complex does not inhibit the phosphatase activity unless subjected to a boiling step which dissociates the modulator subunit. The unboiled [MX] exhibits the activation as well as the inactivation characteristics of the free modulator.     

Complete primary structure of protein phosphatase inhibitor-1 from rabbit skeletal muscle

The complete primary structure of protein phosphatase inhibitor-1 has been determined. The protein consists of a single polypeptide chain of 165 residues, molecular weight 18640. The threonine residue that must be phosphorylated for activation is at position 35 and the active cyanogen bromide peptide, CB-1, comprises residues 2-66. The N-terminal methionine is acetylated and 40% of the inhibitor-1 molecules lack the C-terminal dipeptide Ala-Val. Serine-67 is substantially phosphorylated in vivo, but this phosphoserine residue does not appear to influence the activity of inhibitor-1.     

Identification and partial characterization of a latent ATP,Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase F_A-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M _r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF Phenylmethanesulphonyl Fluoride - TLCK L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride - TPCK L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone     

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeletal muscle

Summary A heat- and acid-stable proten inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does not occur when phosphorylated histone or phosphorylated protamine are used as substrates.     

Isolation of alpha-connectin, an elastic protein, from rabbit skeletal muscle

alpha-Connectin (also called titin 1) has been isolated from rabbit back muscle. Myofibrils were well washed with 5 mM NaHCO3 and then extracted with 0.2 M sodium phosphate, pH 7.0. The extract was dialyzed against 0.1 M potassium phosphate, pH 7.0, to sediment myosin. The supernatant, adjusted to 0.18 M potassium phosphate, pH 7.0, and 4 M urea, was subjected to DEAE Toyopearl column chromatography. beta-Connectin was eluted in the flow-through fraction and alpha-connectin was eluted at around 0.1 M NaCl, when a 0 to 0.25 M NaCl gradient was applied. The separated alpha-connectin was dialyzed against 0.2 M potassium phosphate, pH 7.0. The resultant alpha-connectin showed the same mobility as that in an SDS extract of rabbit back muscle on SDS gel electrophoresis using 1.8% polyacrylamide gels. A monoclonal antibody against chicken breast muscle beta-connectin reacted with the alpha-connectin isolated from rabbit back muscle.     

Isolation and characterization of a high molecular weight glycoprotein from human blood platelets.

A high molecular weight glycoprotein consisting of three disulfide-linked 142,000 molecular weight chains has been isolated from human blood platelets. The glycoprotein, designated thrombospondin, is released by platelets in response to thrombin treatment and is proteolyzed when left in the presence of platelets after liberation. It is relatively insensitive to degradation by thrombin. Thrombospondin is a filamentous protein of dimensions approximately 7 X 70 nm and contains 1.9% neutral sugars, 1.4% amino sugars, 0.7% sialic acid, and no hexuronic acid. Amino acid analysis reveals that the level of cysteine is approximately 260 residues per molecule. Thrombospondin binds to immobilized heparin but is released by 0.45 M sodium chloride. A single band is obtained by isoelectric focusing, indicating a pI of 4.7 as well as a relatively high degree of purity. Degradation of the intact molecule with trypsin yields a stable core particle of molecular weight 210,000 comprised of three 70,000 chains.