An enzyme-probe study of motile domains in the subfragment-2 region of myosin

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.     

Aminoacyl-tRNA synthetases: inter-site interaction as a possible proofreading mechanism

Smooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using ³¹P-NMR. In relaxed muscle obtained in calcium-free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics.     

Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis

Acetate supplementation in rats increases plasma acetate and brain acetyl-CoA levels. Although acetate is used as a marker to study glial energy metabolism, the effect that acetate supplementation has on normal brain energy stores has not been quantified. To determine the effect(s) that an increase in acetyl-CoA levels has on brain energy metabolism, we measured brain nucleotide, phosphagen and glycogen levels, and quantified cardiolipin content and mitochondrial number in rats subjected to acetate supplementation. Acetate supplementation was induced with glyceryl triacetate (GTA) by oral gavage (6 g/kg body weight). Rats used for biochemical analysis were euthanized using head-focused microwave irradiation at 2, and 4 h following treatment to immediately stop metabolism. We found that acetate did not alter brain ATP, ADP, NAD, GTP levels, or the energy charge ratio [ECR, (ATP + ½ ADP)/(ATP + ADP + AMP)] when compared to controls. However, after 4 h of treatment brain phosphocreatine levels were significantly elevated with a concomitant reduction in AMP levels with no change in glycogen levels. In parallel studies where rats were treated with GTA for 28 days, we found that acetate did not alter brain glycogen and mitochondrial biogenesis as determined by measuring brain cardiolipin content, the fatty acid composition of cardiolipin and using quantitative ultra-structural analysis to determine mitochondrial density/unit area of cytoplasm in hippocampal CA3 neurons. Collectively, these data suggest that an increase in brain acetyl-CoA levels by acetate supplementation does increase brain energy stores however it has no effect on brain glycogen and neuronal mitochondrial biogenesis.     

Alterations in myocardial energy metabolism induced by the anti-cancer drug doxorubicin

Doxorubicin and other anthracyclines are among the most potent chemotherapeutic drugs for the treatment of acute leukaemia, lymphomas and different types of solid tumours such as breast, liver and lung cancers. Their clinical use is, however, limited by the risk of severe cardiotoxicity, which can lead to irreversible congestive heart failure. There is increasing evidence that essential components of myocardial energy metabolism are among the highly sensitive and early targets of doxorubicin-induced damage. Here we review doxorubicin-induced detrimental changes in cardiac energetics, with an emphasis on the emerging importance of defects in energy-transferring and -signalling systems, like creatine kinase and AMP-activated protein kinase.     

Skeletal muscle: Energy metabolism, fiber types, fatigue and adaptability

Skeletal muscles cope with a large range of activities, from being able to support the body weight during long periods of upright standing to perform explosive movements in response to an unexpected threat. This requires systems for energy metabolism that can provide energy during long periods of moderately increased energy consumption as well as being able to rapidly increasing the rate of energy production more than 100-fold in response to explosive contractions. In this short review we discuss how muscles can deal with these divergent demands. We first outline the major energy metabolism pathways in skeletal muscle. Next we describe metabolic differences between different muscle fiber types. Contractile performance declines during intense activation, i.e. fatigue develops, and we discuss likely underlying mechanisms. Finally, we discuss the ability of muscle fibers to adapt to altered demands, and mechanisms behind these adaptations. The accumulated experimental evidence forces us to conclude that most aspects of energy metabolism involve multiple and overlapping signaling pathways, which indicates that the control of energy metabolism is too important to depend on one single molecule or mechanism.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

Creatine kinase kinetics,ATP turnover,and cardiac performance in hearts depleted of creatine with the substrate analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% of the creatine substrate analogue β-guanidinopropionic acid for 6–10 weeks. ³¹P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 μmol/g dry wt in guanidinopropionic acid-fed animals but no change in [P_i], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine → ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 μmol/g dry wt per s in control hearts and decreased 4-fold to 2.5–2.8 μmol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2–7.8 μmol/g dry per s. These rates are 1.5–3-times greater than the rate of the phosphocreatine → ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.     

Quantitative assessment of neurochemical changes in a rat model of long-term alcohol consumption as detected by in vivo and ex vivo proton nuclear magnetic resonance spectroscopy

The aim of present study was to quantitatively investigate the neurochemical profile of the frontal cortex region in a rat model of long-term alcohol consumption, by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) at 4.7 T and ex vivo¹H high-resolution magic angle spinning (HR-MAS) technique at 11.7 T. Twenty male rats were divided into two groups and fed a liquid diet for 10 weeks. After 10 weeks, in vivo¹H MRS spectra were acquired from the frontal cortex brain region. After in vivo¹H MRS experiments, all animals were sacrificed and 20 frontal cortex tissue samples were harvested. All tissue examinations were performed with the 11.7 T HR-MAS spectrometer and high-resolution spectra were acquired. The in vivo and ex vivo spectra were quantified as absolute metabolite concentrations and normalized ratios of total signal-intensity (i.e., metabolites_Norm), respectively. The absolute quantifications of in vivo spectra showed significantly higher glycerophosphocholine plus phosphocholine (GPC + PCh) and lower myo-inositol (mIns) concentrations in ethanol-treated rats compared to controls. The quantifications of ex vivo spectra showed significantly higher PCh_Norm, Cho_Norm and tCho_Norm, and lower GPC_Norm and mIns_Norm ratio levels in ethanol-treated rats compared to controls. Our findings suggest that reduced mIns concentrations caused by the long-term alcohol consumption may lead to hypo-osmolarity syndrome and astrocyte hyponatremia. In addition, increased choline-containing compound concentrations may reflect an increased cell turnover rate of phosphatidylcholine and other phospholipids, indicating an adaptive mechanism. Therefore, these results might be utilized as key markers in chronic alcohol intoxication metabolism.     

Whole body exposure to low frequency magnetic field: No provable effects on the cellular energetics of rat skeletal muscle

On the basis of previous experience with biological effects of electromagnetic fields a potential effect of homogeneous sinusoidal magnetic field (50Hz, 10mT) on energy state of rat skeletal muscle was investigated. Two different total body exposures to magnetic field were selected: (1) repeated 1 hour exposure, 2 times a week for 3 months, and (2) acute 1.5 hour exposure (and the appropriate control groups). Important energy metabolites (adenosine triphosphate – ATP, creatine phosphate, creatine, lactate, pyruvate and inorganic phosphate) were analysed by enzymatic and spectroscopic methods in musculus gracilis cranialis.On the basis of the concentration of important energy metabolites the apparent Gibbs free energy of ATP hydrolysis and creatine charge was calculated. Our results demonstrate no influence of this low frequency magnetic field on the level of important energy metabolites in rat skeletal muscle. The conclusion of this study is that neither repeated exposure nor the acute exposure of rats to the sinusoidal magnetic field of given parameters has any important influence on the energy state of the skeletal muscle.     

Mixed macromolecular crowding accelerates the refolding of rabbit muscle creatine kinase: implications for protein folding in physiological environments

The effects of four single macromolecular crowding agents, Ficoll 70, dextran 70, polyethylene glycol (PEG) 2000, and calf thymus DNA (CT DNA), and three mixed crowding agents containing both CT DNA and polysaccharide (or PEG 2000) on the refolding of guanidine hydrochloride-denatured rabbit muscle creatine kinase (MM-CK) have been examined by activity assay. When the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to Ficoll 70 is 1:9, the refolding yield of MM-CK after refolding for 3 h under these conditions increases 23% compared with that in the presence of 10 g/l CT DNA, 18% compared with 100 g/l Ficoll 70, and 19% compared with that in the absence of crowding agents. A remarkable increase in the refolding yield of MM-CK by a mixed crowding agent containing CT DNA and dextran 70 (or PEG 2000) is also observed. Further folding kinetics analyses show that these three mixed crowding agents remarkably accelerate the refolding of MM-CK, compared with single crowding agents. Aggregation of MM-CK in the presence of any of the three mixed crowding agents is less serious than that in the presence of a single crowding agent at the same concentration but more serious than that in the absence of crowding agents. Both the refolding yield and the refolding rate of MM-CK in mixtures of these agents are increased relative to the individual agents by themselves, indicating that mixed macromolecular crowding agents are more favorable to MM-CK folding and can be used to reflect the physiological environment more accurately than single crowding agents.