Isolation and properties of lysyl-tRNA-synthetase from rabbit skeletal muscles

A method is developed to isolate lysyl-tRNA-synthetase from 93-95%-purity postribosomal supernatant fraction of skeletal muscle homogenate in rabbit. Novelty of the method is the ATP usage for muscle homogenization, which permits shortening the number of operations during the enzyme isolation. The molecular weight of protein is 68 +/- 10 kDa, the monomer unit consists of 540 amino acids and contains a carbohydrate component.     

Metabolic and contractile properties of rabbit muscles: a statistical approach

The metabolic and contractile properties of rabbit muscles representative of the three main muscle types have been studied. A statistical analysis of the results indicates the more discriminant variables for this characterization and shows in particular that the myosin light chains represent a very significant discriminant factor of the muscular type. Results also show that lactate dehydrogenase activity is strongly correlated with the percentage of lactate dehydrogenase M-subunit and with myosin light chains.     

Immobilized active monomers of D-glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscles and their coenzyme-binding properties

Experimental conditions favouring the dissociation of tetrameric rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase into active monomers were elaborated. The urea-induced dissociation of the tetramer was shown to be a stepwise process (in 2 M urea only dimers are formed; an increase in urea concentration up to 3 M causes the splitting of the dimers into monomers). The specific activity of immobilized monomers in the glyceraldehyde-3-phosphate oxidation reaction does not differ from that of the parent immobilized tetrameric form. The tetrameric enzyme molecule binds the coenzyme with a negative cooperativity (the first two NAD+ molecules bind with KD below 0.1 microM; for the third and fourth molecules the dissociation constant was determined to be equal to 5.5 +/- 1.5 microM (50 mM medinal buffer, 10 mM sodium phosphate, pH 8.2). The cooperativity of NAD+ binding is preserved in the immobilized preparation of tetrameric dehydrogenase. The immobilized monomers bind NAD+ with KD of 1.6 +/- 1.0 microM. The experimental results are consistent with the hypothesis according to which the association of catalytically active subunits into a tetramer changes their coenzyme-binding properties in such a way that the first two NAD+ molecules bind more firmly to a tetramer than to a monomer, whereas the third and the fourth NAD+ molecules bind less firmly.     

Chronic strain alters the passive and contractile properties of rabbit airways.

Pathophysiological conditions of the lung may shift the balance of forces so as to chronically alter the amount of strain imposed on the airways. This chronic strain could result in changes in the structure and/or function of the airways that affect its physiological properties. We evaluated the effects of imposing physiological levels of chronic mechanical strain on the passive and active physiological properties of intraparenchymal rabbit airways. Isolated bronchial segments were cultured for 48 h at transmural pressures of 0 cmH(2)O (No Strain) or 7 cmH(2)O (Strain). Effects of strain on small parenchymal airways were evaluated in lung tissue slices cultured under conditions of No Strain or approximately 50% increased in diameter (Strain). Chronic strain resulted in a higher passive compliance of the bronchial segments and larger airway lumen size. In addition, bronchi not subjected to chronic Strain were more responsive to ACh than bronchi subjected to chronic Strain, and airways in lung slices subjected to No Strain narrowed more in response to ACh than airways in lung slices subjected to Strain. The greatest effects of chronic strain occurred in the smallest sized airways. Our results suggest that chronic distension of the airways has physiologically important effects on their passive and active properties, which are most prominent in the smaller, more peripheral airways.     

Comparative studies of contractile proteins from the skeletal and cardiac muscles of lower vertebrates

The available evidence suggests that the properties of the contractile proteins from lower vertebrates are broadly similar to those found in skeletal and cardiac muscles of mammalian species. However, the proteins from ectotherms are generally more unstable on isolation.     

The role of passive structures in force enhancement of skeletal muscles following active stretch

We recently found that force enhancement following active stretch in skeletal muscles is accompanied by an increase in passive force following deactivation (J. Exp. Biol. 205 (2002) 1275). However, it is not known if this increase in passive force contributes to the force enhancement observed in the active muscle, and if it is observed at all muscle lengths. The purposes of this study were to quantify the amount of passive force increase as a function of muscle lengths, and to determine if this passive force contributes to the force enhancement observed in the active muscle. Experiments were performed on cat soleus (n = 24) using techniques published previously (J. Biomech. 30(9) (1997) 865). Conceptually, tests involved comparisons of force enhancement and passive force increase for a variety of stretch tests in soleus. Furthermore, in one test, activation of the soleus was interrupted for 1s in the force-enhanced state, and soleus was then re-activated. We found that total force enhancement and passive force increase were positively correlated for all test conditions, that passive force increase following stretch of the active soleus only occurred at muscle lengths corresponding to the descending limb of the force-length relationship, that increases in passive force for a given stretch magnitude became greater at long muscle lengths, and that upon reactivation, there was a remnant passive force enhancement. We conclude from these results that the passive force enhancement following stretch of an active muscle contributes to the total force enhancement, that this passive contribution increases with increasing muscle length, and that there must be at least one other factor than passive force increase that contributes to the total force enhancement, as the passive force increase was always smaller than the total force enhancement. A by-product of this investigation was that we observed a shift in the passive force-length relationship that was dependent on muscle activation, stretch magnitude and muscle length. Therefore, the passive force-length relationship is not a constant property of skeletal muscle, but depends critically on the muscle's contractile history.     

Changes in structural and functional properties of actin in denervated rabbit skeletal muscles

Amino acid composition of actin from white skeletal muscles of the rabbit was studied 14 and 50 days after denervation. The content of valine, histidine and tyrosine was shown to decrease and that of lysine and leucine to increase on the 14th day following denervation. The content of these amino acids was almost normalised on the 50th day. In case of actin obtained from the contralateral leg similar but less pronounced changes were revealed. The results obtained are discussed as to the role played by some of amino acids in actin polymerization.     

Modification of the contractile properties of rabbit skeletal muscle by ethylene glycol

The effect of ethylene glycol on the contractile properties of skeletal muscles was studied using glycerinated rabbit psoas muscle fibers. Measurements were made at an ionic strength of 0.2 M, pH 7.0, and at 10 degrees C. Ethylene glycol reversibly reduced isometric tension, active stiffness, the tension-to-stiffness ratio, and the shortening velocity at zero load (Vo) in a dose-dependent fashion. Ethylene glycol also reduced the Ca sensitivity for contraction. The extent of the reduction in Vo by ethylene glycol was much larger than that in the actomyosin ATPase activity reported by Travers and Hillaire (Eur. J. Biochem. 98, 293-299 [1979]). Although ethylene glycol reduced tension and Vo, the MgATP concentration dependence of these two quantities was almost unaffected. These results suggest that in the presence of ethylene glycol, force produced by crossbridges in the principal force-producing state is reduced and/or the relative population of the attached crossbridges in the low-force state increases. The results also suggest that the reduction in Vo by ethylene glycol is caused not only by a reduction in the actomyosin ATPase activity but also by a reduction in the shortening distance per mole of ATP split.     

Interaction of phosphorylase kinase with thin filament proteins of rabbit skeletal muscles

The binding of phosphorylase kinase to thin filaments and their effects on the enzyme activity as well as the contribution of the enzyme to contractile protein phosphorylation have been studied. The data obtained suggest that the kinase binding to thin filaments is controlled by the regulatory proteins, troponin and tropomyosin. The bulk of the enzyme is bound to the F-actin-tropomyosin-troponin complex which activates the enzyme in a far greater degree than each of its constituent components. Ca2+ and ATP control the kinase binding to F-actin. ATP increases the enzyme binding 6-fold; Ca2+ decrease the S0.5 value for F-actin 5-fold. In acetone powder extracts phosphorylase kinase phosphorylates thin filament-bound phosphorylase b, troponin T and troponin I as well as 51-58 kDa and 114 kDa proteins. These results suggest that phosphorylase kinase plays a role in the mechanism of synchronization of glycogenolysis and muscle contraction rates.     

Nitric oxide release and contractile properties of skeletal muscles from mice deficient in type III NOS

Skeletal muscle constitutively expresses both the type I (neuronal) and type III (endothelial) isoforms of nitric oxide synthase (NOS). We tested the functional importance of type III NOS using skeletal muscles with similar levels of type III NOS expression (diaphragm and soleus) from wild-type, heterozygous, and type III NOS-deficient littermate mice. Muscles were incubated at 37 degrees C in Krebs-Ringer solution. NO accumulation in the medium was measured by chemiluminescence; force-frequency and fatigue characteristics were measured using direct electrical stimulation. Diaphragm and soleus released NO at similar rates during passive incubation; these rates increased during active contraction. NO release by type III NOS-deficient muscle was not different from that of wild-type muscle under any condition tested. Force-frequency and fatigue characteristics also were unaffected by genotype. Because type III NOS deficiency did not alter function, we conclude that NO effects previously observed in wild-type muscle are likely to be mediated by type I NOS.     

Subunit composition and enzymatic properties of myosins from human and rabbit skeletal muscles

It is shown that myosin of human skeletal muscles is more difficult for purification from the actin and nucleic acids admixtures. It is also characterized by a less yield and a pronounced lability to denaturant effects as compared to rabbit myosin. The ATPase activity of human myosin is 1.5-2 times as low and the cholinesterase one--tens of times as high as those of rabbit myosin. A relative content of LC3 (LC--light chains) is approximately twice as low and that of LC1--as high as in rabbit myosin. It is supposed that the found differences in the properties may be explained to a considerable extent by a different ratio of certain light chains contained in the investigated proteins.     

Essential carboxylic groups of active sites of creatine kinase M- and M'-subunits in rabbit skeletal muscles

The influence of carboxylic group modification with N-cyclohexyl-N'-beta-(4-methyl-morpholine)ethylcarbodiimide on the activity of creatine kinase was examined. The modification rate for M- and M'-subunits of the enzyme depends on the reagent concentration and the presence of Mg2+ ions. The process is described by linear dependences of logarithms of activity vs. time, indicating a pseudofirst order of reactions. The reagent inactivates M- and M'-subunits of the enzyme at approximately the same rate, modification being characterized by rate constants of 0.17 min-1 and K1 of 0.17 M. Mild alkaline hydrolysis (pH 9.2) of the modified enzyme leads to partial (30-60%) restoration of its activity. The addition of [14C]glycine methyl ester results in the irreversible incorporation of radioactivity into the protein. AMP and ATP gamma-(p-azido-anilide), the inhibitors acting competitively to nucleotide substrates, protect the enzyme against inactivation with the reagent, whereas creatine and creatine phosphate exert no influence on the modification rate of the enzyme. It is suggested that modification affects the aspartate or glutamate carboxyls at or near the ATP-binding sites in the M- and M'-subunits of the enzyme.     

Ciliary neurotrophic factor (CNTF) affects the excitable and contractile properties of innervated skeletal muscles

The well-established trophic role of CNTF upon neurons led to performing clinical trials in patients of neurodegenerative diseases. However, trials were suspended due to side effects such as severe weight loss, hyperalgesia, coughing, muscle cramps and pain. So far it is not known how CNTF triggers the problems related to skeletal muscle cramps and pain. CNTF has also been described as a myotrophic factor for denervated skeletal muscles, but the possibility that it affects innervated muscles has also been considered. Since a myotrophic factor could be a valuable tool for treatment of several muscle diseases, we studied the effects of low doses of CNTF delivered systemically by an osmotic pump, over the electrical and mechanical properties of innervated and denervated fast and slow muscles. CNTF induced spontaneous electrical discharges and slowed twitches in innervated muscles, but did not prevent the changes induced by denervation. We postulate that the spontaneous discharges induced by CNTF in innervated muscles may be the cause of the cramps, coughing, and muscle ache reported by patients. At low doses, CNTF does not exert its myotrophic role over denervated muscles but clearly affects the excitable and contractile properties of innervated muscles.     

Orientation of tendons in vivo with active and passive knee muscles

Tendon orientations in knee models are often taken from cadaver studies. The aim of this study was to investigate the effect of muscle activation on tendon orientation in vivo. Magnetic resonance imaging (MRI) images of the knee were made during relaxation and isometric knee extensions and flexions with 0 degrees , 15 degrees and 30 degrees of knee joint flexion. For six tendons, the orientation angles in sagittal and frontal plane were calculated. In the sagittal plane, muscle activation pulled the patellar tendon to a more vertical orientation and the semitendinosus and sartorius tendons to a more posterior orientation. In the frontal plane, the semitendinosus had a less lateral orientation, the biceps femoris a more medial orientation and the patellar tendon less medial orientation in loaded compared to unloaded conditions. The knee joint angle also influenced the tendon orientations. In the sagittal plane, the patellar tendon had a more anterior orientation near full extension and the biceps femoris had an anterior orientation with 0 degrees and 15 degrees flexions and neutral with 30 degrees flexions. Within 0 degrees to 30 degrees of flexion, the biceps femoris cannot produce a posterior shear force and the anterior angle of the patellar tendon is always larger than the hamstring tendons. Therefore, co-contraction of the hamstring and quadriceps is unlikely to reduce anterior shear forces in knee angles up to 30 degrees . Finally, inter-individual variation in tendon angles was large. This suggests that the amount of shear force produced and the potential to counteract shear forces by co-contraction is subject-specific.