Spontaneous fluctuations in the activity of rabbit skeletal muscle NAD-kinase]

Spontaneous fluctuations in the time of the activity of the 280-300-fold purified NAD-kinase preparation from rabbit skeletal muscle following its dilution are described. Defrosted but undiluted enzyme preparation failed to exhibit any fluctuations in its activity.     

The effect of carnosine on Ca-channels in rabbit skeletal muscle sarcoplasmic reticulum]

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.     

Several properties of the Ca-pump of rabbit skeletal muscle sarcoplasmic reticulum in hypercholesteremia]

Sarcoplasmic reticulum (SR) fragments from the skeletal muscles of rabbit with marked atherosclerosis possessed decreased Ca2+-accumulating capacity. Lowering of transport efficiency, namely reduction of the Ca/ATP ratio from 1.9--normal value--to 0.9 during the experiment at 26 degrees C was accompanied by activation of Ca-ATPase and simultaneously of the rate of Ca2+ outflux from the SR. Arrhenius plots of Ca-ATPase temperature dependence characterized under normal conditions by a break at 20--21 degrees C was linearized under hypercholesterolemia. At the same time there was a rise (from 0.03 under normal conditions to 0.15 in atherosclerosis) of cholesterol/protein ratio in the SR membrane preparations. Activation energy for Ca-ATPase crude membranes under normal conditions was equal to 15.6 and 28.7 kcal/mol above and below the break point respectively; this value for Ca-ATPase of membranes with increased cholesterol level was 19 kcal/mol for all the temperatures investigated.     

Isolation and characterization of glycogen branching enzyme from rabbit liver

Glycogen branching enzyme was isolated from rabbit liver. The highly purified enzyme shows a monomer molecular weight of 71 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and apparent molecular weights of 93 000 by sucrose density gradient sedimentation and 52 000 by gel-exclusion chromatography on Sephacryl S-300. No glucosamine, mannosamine, galactosamine, or sialic acid was detected in the protein. An amino acid analysis is reported. The spectrum of branching enzyme is that of a simple polypeptide, with A1%280nm = 24.6. Highly purified branching enzyme consists of several closely related active enzyme forms that can be resolved by isoelectric focusing in polyacrylamide gel. The major species of pI 5.7 is flanked by less abundant forms of pI 5.6 and 5.8. Seemingly identical enzyme forms are observed in crude extracts of rabbit liver, skeletal muscle, brain, and heart, although the absolute and relative concentrations vary among the tissues. Branching enzyme apparently does not exhibit tissue-specific isoenzymes.     

Structural and functional properties of Drosophila melanogaster phosphorylase: comparison with the rabbit skeletal muscle enzyme

Glycogen phosphorylase isolated from Drosophila melanogaster contains one pyridoxal 5'-phosphate per subunit; the coenzyme is in a hydrophobic environment. Fruit-fly phosphorylase a has lower KM for glucose-1-phosphate and is less sensitive to allosteric inhibitors than the b form of the enzyme. The amino acid composition of Drosophila phosphorylase differs from that of rabbit skeletal muscle phosphorylase. These two enzymes give distinct one dimensional peptide maps. The distribution of reactive SH-groups is markedly different in the insect and vertebrate phosphorylase. Fruit-fly phosphorylase a is dephosphorylated by either rabbit or Drosophila protein phosphatase-1 at a slower rate than rabbit muscle phosphorylase a.     

Effect of SH-reagents on ATPase systems of rabbit skeletal muscle nuclei]

The influence of sulfhydryl reagents on ATPase systems of rabbit sceletal muscles nuclei was studied. It is found that p-ChMB at low concentration similarly inhibits both Mg2+- and Mg2+, Ca2+-ATPases. p-ChMB at higher concentrations inhibits completely Mg2+, Ca2+-ATPase, while Mg2+- ATPase--only by 60%. N-EM is lesser specific inhibitor of SH-groups, than p-ChMB. The degree of nuclear ATPases inhibition by N-EM is practically identical. Using inhibitory analysis, two hypes of skeletal muscles nuclei SH-groups are found: easily reacting with N-EM, and those reacting with N-EM at more high concentrations, which are essential for ATPase ATP-hydrolysing activity. ATP defends Mg2+, Ca2+-ATPase, but not the Mg2+-ATPase from N-EM inhibitory action. Cysteine completely eliminates the inhibitory effect of p-ChMB on Mg2+-ATPase but only 40% on MG2+, Ca2+-ATPase. Mg2+, Ca2+-ATPase of nuclei is more sensitive to the sulfhydryl venoms action than Mg2+-ATPase.     

The existence of an insoluble Z disc scaffold in chicken skeletal muscle

Extraction of glycerinated chicken skeletal muscle with 0.6 M potassium iodide leaves a framework of insoluble components within each muscle fiber. This framework is composed primarily of planes of in-register Z discs that have been thickened by the accumulation of material on both sides of each disc during extraction. Membrane vesicles, presumably remnants of the T system, remain surrounding the Z discs. When the framework is sheared in a blender, it is preferentially cleaved between Z planes, resulting in the formation of large sheets of interconnected, closely packed Z discs in a honeycomb-like array. Cleavage occurs in regions formerly occupied by the A bands, which have been weakened by the removal of myosin. The existence and stability of these planar Z disc arrays demonstrate the presence and strength of connections between adjacent myofibrils.SDS-polyacrylamide gel electrophoresis reveals that this framework consists primarily of actin and desmin, with lesser amounts of a few proteins including α-actinin, myosin and tropomyosin. Z disc sheets and KI-extracted myofibrils provide a distinct face-on view and side view, respectively, of the Z disc. In indirect immunofluorescence, these two views have revealed that desmin is present at the periphery of each Z disc, forming a network of proteinaceous collars within the Z plane. α-Actinin is localized within each disc, giving a face-on fluorescence pattern that is complementary to that of desmin. Actin is present throughout the thickened Z plane, while myosin and tropomyosin exist only in the insoluble residue that coalesces on both faces of each disc.We conclude that desmin, perhaps in conjunction with actin, is responsible for interlinking Z discs of adjacent myofibrils, and may thus serve as a mechanical and structural integrator of muscle fibers. Its hydrophobic nature and coincident distribution with the T system suggest that it may also be responsible for mediating filament-membrane interactions and anchoring the triad to the Z disc. Its collar-like distribution suggests that it may aid in maintaining the structural integrity of the Z disc and the actin filaments inserted into it.     

The immunohistochemical location of cathepsin L in rabbit skeletal muscle

Summary The immunohistochemical location of cathepsin L in rabbit soleus, plantaris and psoas muscles was investigated using the peroxidase-anti-peroxidase (PAP) technique. The amount of enzyme detected varied according to the fibre type, which were identified by histochemical staining of serial sections for succinate dehydrogenase and alkali-stable myosin ATPase. In the three muscles studied labelling was strongest in the highly oxidative fibres and weaker in the other fibre types with least staining in the fast white fibres. Immunoreactive cathepsin L appeared to be most concentrated at the periphery of muscle fibres, especially near to the nuclei, although some staining was seen throughout the fibres.     

The amino acid sequence of rabbit skeletal muscle glycogenin

The amino acid sequence of glycogenin from rabbit skeletal muscle has been determined. The N-acetylated protein consists of 332 amino acids and has a molecular mass of 37278 Da. The novel tyrosyl-glucose linkage between glycogenin and glycogen [Smythe, C., Caudwell, F. B., Ferguson, M. & Cohen, P. (1988) EMBO J. 7, 2681-2686] is shown to occur at a single site, tyrosine-194. Although glycogenin is a UDP-Glc utilising glucosyltransferase that self-glucosylates [Pitcher, J., Smythe, C. & Cohen, P. (1988) Eur. J. Biochem. 176, 391-395], following addition by an unknown enzyme of the first glucose to tyrosine-194, it is not homologous to either human glycogen synthase or other UDP-Glc-requiring enzymes.