Phosphorylation of skeletal muscle microsomes by acetylphosphate

The phosporyl group of acetylphosphate is transferred to the membranal protein of the sarcoplasmic vesicles during active calcium transport. Although the phosphoprotein formed cannot be distinguished from that obtained in the presence of ATP, the conditions for ATP and acetylphosphate hydrolysis are different from each other.     

Inhibition by alanine of AMP-deaminase from rabbit skeletal muscle.

1. Alanine inhibits rabbit muscle AMP-deaminase while aspartate, histidine and glutamate are ineffective. 2. The degree and type of inhibition of AMP-deaminase by alanine depend on pH; at pH 6.5 alanine behaves like an allosteric effector exerting a negative heterotropic effect. At pH 7.0 the inhibition is non-competitive, Ki being as high as 19 mM. 3. The probable significance of the effect of alanine on AMP-deaminase in muscle metabolism is discussed.     

Binding of myosin cross-bridges to thin filaments of rabbit skeletal muscle.

Cross-linking of myosin subfragment 1 (S1) with a molar excess of actin in vitro reveals the presence of an actin-S1-actin complex. It is absolutely essential that actin be present in molar excess over S1 so that the decoration of F-actin with S1 be incomplete. However, the excess of actin may not be available in the overlap zone of sarcomeres of skeletal muscle. We therefore found it necessary to test for the presence of the actin-S1-actin complex in vivo. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the products were resolved by polyacrylamide gel electrophoresis and analyzed by Western blots using antibodies against actin and against heavy and light chains of myosin. The cross-linking produced the evidence of formation of actin-S1-actin complex.     

Cysteine proteinase inhibitors from rabbit skeletal muscle

1. Two cysteine proteinase inhibitors, I-T (Mr = 29,000) and I-S (Mr = 10,700), were isolated from rabbit skeletal muscle by means of succesive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75. 2. I-T is a formed trimer of a monomeric inhibitor, I-M (Mr = 10,500), through disulfide bonds. 3. I-S is almost completely stable between pH 3 and 8, while I-M is unstable in the same pH range. 4. I-M acts most effectively towards cathepsins H and L, showing moderate activity towards cathepsin B and only weak activity towards papain. I-S acts most effectively towards cathepsin L, followed by, in decreasing order, cathepsin H, cathepsin B and papain.     

Phosphorylation of K-casein by glycogen synthase kinase-3 from rabbit skeletal muscle

Glycogen synthase kinase-3 (ATP:protein phosphotransferase, EC 2.7.1.37) phosphorylated K-casein 20-fold more rapidly than beta-casein, while alpha S1-casein was not a substrate. This distinguished it from casein kinase-I and casein kinase-II, which phosphorylate the beta-casein variant preferentially. Glycogen synthase kinase-3 phosphorylated a serine residue(s) in the C-terminal cyanogen bromide fragment on K-casein. In contrast, cyclic AMP-dependent protein kinase phosphorylated the N-terminal fragment, and phosphorylase kinase the N-terminal and intermediate cyanogen bromide fragments. The results emphasize the potential value of casein phosphorylation as a means of classifying protein kinases.     

Amino acid sequence of acylphosphatase from rabbit skeletal muscle

The complete amino acid sequence of acylphosphatase from rabbit skeletal muscle has been elucidated by automatic Edman degradation of peptides obtained from staphylococcal protease and trypsin digestions. The enzyme consisted of a single polypeptide chain of 98 amino acid residues, lacking only histidine. Its amino (N)-terminus was blocked by an acetyl group. The presented sequence of rabbit muscle enzyme was compared with those of equine and porcine muscle enzymes. There were four unique replacements, i.e., Arg-4, Asp-28, Arg-31, and Glu-56 in the sequences of both equine and porcine muscle enzymes were replaced by Gly, Gly, Lys, and Asp, respectively, in that of rabbit muscle enzyme. Extensive structural homology was observed among the three enzymes.     

Domain organization of alpha-actinin from the rabbit skeletal muscle

Limited trypsinolysis of native alpha-actinin by trypsin was carried out. This procedure resulted in the formation of two large fragments with Mr of 30 and 70 kD which cover almost the whole subunit of alpha-actinin. Using the sedimentation equilibrium method, it was demonstrated that the bisubunit structure of alpha-actinin is provided for by C-terminal fragments of the subunits. Data from circular dichroism analysis suggest that the fragments formed are independent structural units, i.e., domains.     

Cooperative self-association of phosphorylase kinase from rabbit skeletal muscle

Ca(2+)- and Mg(2+)-induced association of phosphorylase kinase (PhK) from rabbit skeletal muscle has been studied at the magnitudes of the ionic strength close to the physiological values (40 mM Hepes, pH 6.8, containing 0.1 M NaCl, 0.1 mM Ca(2+), 10 mM Mg(2+); 25 degrees C) and under the molecular crowding conditions produced by high concentrations (1 M) of the natural osmolyte, trimethylamine N-oxide (TMAO). In the presence of 0.1 M NaCl two forms of PhK were registered, namely the "basic form" and "highly associated form", suggesting that PhK association may be treated as an example of cooperative association. According to the data on dynamic light scattering the average hydrodynamic radii of these forms were 16 and 144 nm. The addition of 1 M TMAO produces the time dependent increase in the light scattering intensity caused by the conversion of the basic form into the highly associated form. According to the data of the sedimentation analysis the basic form of PhK comprises a hexadecamer (M(r)=1320 kDa) and its small associates. The removal of Ca(2+) by addition of EGTA results in the reverse conversion of the highly associated form into the basic form suggesting reversibility of self-association of PhK. FAD, the ligand that is specifically bound to PhK, blocks the conversion of the basic form of PhK into the highly associated form.     

Oxalate dependence of calcium uptake kinetics of rabbit skeletal muscle microsomes (fragmented sarcoplasmic reticulum)

The initial rate of oxalate-facilitated Ca²⁺ uptake by skeletal microsomes depends on both Ca²⁺ and oxalate concentrations in the medium. The apparent K_m for Ca²⁺ increases with increasing oxalate concentration, indicating that Ca²⁺ uptake can involve a carrier-mediated transport system.     

Autophosphorylation of glyceraldehydephosphate dehydrogenase and phosphorylation of protein from skeletal muscle microsomes

Glyceraldehydephosphate dehydrogenase purified from rabbit skeletal muscle is auto-phosphorylated with MgATP. Half-maximal phosphorylation is achieved around 0.3 mM. The phosphorylation is Ca2+ independent. The phosphoenzyme complex is labile in alkaline conditions and stable in moderately acid media. The complex is readily hydrolyzed by 0.1 M neutral hydroxylamine, indicating the complex formed is a high-energy acyl phosphate. The phosphorylation is reduced by nicotinamide adenine dinucleotides, reduced form (NADH), glyceraldehyde 3-phosphate, and nicotinamide adenine dinucleotide (NAD+). The enzyme is also dephosphorylated by these metabolites although to a lesser extent by NAD+. Calsequestrin isolated from rabbit skeletal muscle inhibits the phosphorylation of the enzyme. The phosphoenzyme behaves as a kinase catalyzing the phosphorylation of proteins of Mr 80 000 and 72 000 found in the skeletal muscle terminal cisternae/triad preparation. This reaction is enhanced by NADH. The phosphate found in the protein substrate has been shown to be the same phosphate initially involved in the phosphorylation of glyceraldehydephosphate dehydrogenase.     

Block of endplate channels by permeant cations in frog skeletal muscle

Motor endplates of frog semitendinosus muscles were studied under voltage clamp. Current fluctuations induced by iontophoretic application of acetylcholine were analyzed to give the elementary conductance, gamma , and mean open time, tau , of endplate channels. Total replacement of the external Na+ ion by several other metal ions and by many permeant organic cations changed both gamma and tau . Except with NH4+ ions, the gamma values with foreign test ions were all smaller than expected from the independence relation and their previously measured permeability ratios. The more hydrophobic ions gave the smallest gamma values. Foreign permeant cations also depress gamma when mixed with Na+ ions. These effects could be interpreted in terms of binding of ions to a saturable site within the endplate channel as they pass through. The site for organic ions would have a hydrophobic component. Similar evidence is given for a metal ion binding site on the cytoplasmic end of the channel accessible to internal ions. Most foreign cations also shortened tau when applied externally. The changes of gating did not seem to be correlated with changes in gamma . Thus there is no evidence for control of tau by ions bound within the pore.     

Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine

Summary The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (K_i value of 0.3mm) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylasea.     

Phosphorylation of myosin light chain by a protease-activated kinase from rabbit skeletal muscle

A protease-activated protein kinase that phosphorylates the P light chain of myosin in the absence of Ca2+ and calmodulin has been isolated from rabbit skeletal muscle. The enzyme has properties similar to protease-activated kinase I from rabbit reticulocytes [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11588-11564], which has been shown to phosphorylate the P light chain of myosin [P. T. Tuazon, J. T. Stull, and J. A. Traugh (1982) Biochem. Biophys. Res. Commun. 108, 910-917]. The protease-activated kinase from skeletal muscle has been partially purified by chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The enzyme phosphorylates histone as well as the P light chain of myosin following activation by proteolysis. Stoichiometric phosphorylation of myosin light chain was observed with the protease-activated kinase and myosin light chain kinase. The sites phosphorylated by the protease-activated kinase and myosin light chain kinase were examined by two-dimensional peptide mapping following chymotryptic digestion. The phosphopeptides observed with the protease-activated kinase were different from those obtained with the Ca2+-dependent myosin light chain kinase, indicating that the two enzymes phosphorylated different sites on the P light chain of skeletal muscle myosin. When actomyosin from skeletal muscle was examined as substrate, the P light chain was phosphorylated following activation of the protease-activated kinase by limited proteolysis.