Myofibrillar creatine kinase activity inferred from a 3D model.

Myofibrillar creatine kinase (CK) that buffers ATP during fluctuating muscle energy metabolism has been selected for studies of conformational changes underlying the cellular control of enzyme activity. The force field was computed for three energetic states, namely for the substrate-free CK molecule, for the molecule conjugated with the MgATP complex, and for the molecule conjugated with the pair of reactants MgATP-creatine. Without its substrates, the enzyme molecule assumes an inactive "open" form. Upon binding of the MgATP complex, the CK molecule takes up a reactive "closed" conformation. Subsequent binding of creatine yields a nonreactive "intermediary" conformation. Acid-base catalysis is considered to be the basic principle for the reversible transfer of the phosphoryl group between the substrates. The results indicate that the substrate-induced energy minimizing conformational changes do not represent a sufficient condition for CK activity and that some other essential component of physiological control at the cellular level is involved in the transition from the intermediary to the closed structure of the molecule.

Energy metabolism of rat skeletal muscle modulated by the rate of perfusion flow.

In order to advance our understanding of the phenomenon of flow-induced increases in the metabolism of the relaxed muscle, the metabolic rate of the isolated rat gracilis muscle was investigated at 28 degrees C in vitro. The muscle was perfused with cell-free Krebs-Henseleit bicarbonate buffer containing 5% bovine serum albumin and 5 mM glucose, saturated with a gas mixture of 95% O2 and 5% CO2 and simultaneously superfused with a medium saturated with with a low O2 gas mixture (1% O2, 5% CO2 and 94% N2). Two different perfusion flow rates (0.054 and 0.100 ml min-1) have been used. Their influence on oxygen consumption and lactate production has been measured. After a 100 min perfusion period, the muscle was freeze-clamped and analysed for ATP, phosphocreatine, creatine, lactate, pyruvate, inorganic phosphate and glycogen content. The energy state of the cell and the proportions of glycolytic and mitochondrial fluxes of ATP synthesis were evaluated. During perfusion at the low flow rate of 0.054 ml min-1, the oxygen uptake was 45 +/- 9 nmol min-1 (g wet wt)-1, accompanied by a dominance of anaerobic glycolytic synthesis of ATP over mitochondrial ATP synthesis, even though the total delivery of oxygen to muscle was three times higher than oxygen consumption. Increasing the perfusion flow rate to 0.100 ml min-1 increased the oxygen uptake to 120 +/- 6 nmol min-1 (g wet wt)-1, thus leading to a prevalence of mitochondrial ATP synthesis over glycolytic ATP synthesis. The inner stores of glycogen served as the main substrate of energy metabolism and the role of exogenous substrates in the flow-stimulated increase of oxygen uptake was negligible. The increase in perfusion rate also enhanced the energy state of the muscle fibres, which was expressed either as the creatine charge or as the value of the change of Gibbs free energy of ATP hydrolysis. Data indicate that the change of perfusion flow rate per se, apart from oxygen and exogenous substrate supply, elicits changes in the regulation of energy metabolism within non-contracting skeletal muscle under open microcirculation.

Muscle ATP synthesis and utilisation, balanced during flow-induced increase of respiration.

To investigate the control of cell energetic metabolism, creatine charge, ATP/ADP ratio and oxygen consumption (as indicators of an energetic status, the balance between ATP synthesis and degradation and the aerobic ATP turnover, respectively) were evaluated in the rat gracilis muscle, perfused-superfused in vitro. During the perfusion rate of 70 microl/min the ATP/ADP ratio, as well as the creatine charge are kept at the in vivo level. With the decrease of the rate toward 54 microl/min (of an abundant oxygen delivery), the values of both parameters are lower than levels in vivo. With the increase of the rate up to 100 microl/min, both parameters are kept at the in vivo level, when respiration increases by 125%. The data demonstrate the 'unmatched' control of ATP utilisation and synthesis steady rates during the low perfusion rate; during the increasing steady ATP turnover following the increased perfusion rate, the two fluxes are strikingly 'matched', i.e. precisely balanced.

Levels of energy-related metabolites in intact and isolated perfused-superfused rat skeletal muscles.

Adenosine 5'-triphosphate (ATP), phosphocreatine (PCr), creatine (Cr), inorganic phosphate (Pi), lactate (LAC), pyruvate (PYR) and glycogen as glucose (GLU) were determined and free adenosine 5'-diphosphate (ADP) was calculated from ATP:creatine phosphokinase (CPK) reaction in the gracilis muscle of cold-acclimated rats in vivo, and in completely isolated muscles under medium perfusion and superfusion in vitro, using the freeze-clamping method. The mean in vivo levels (mumol/g w.w.) were: ATP 4.8, PCr 12.0, Cr 7.8, Pi 16.1, LAC 1.6, PYR 0.09, GLU 22.9, ADP 0.62 x 10(-3). Isolation of the muscle (about 11 min of anoxia followed by perfusion in the air with a high pO2 medium) decreased macroergic phosphate levels (ATP 3.0, PCr 8.3). In isolated muscles perfused with a high pO2 medium (99 kPa O2, perfusion rate 70 microliters/min) and simultaneously superfused with a low pO2 medium (6.2 kPa O2, 2.3 ml/min) at 28 degrees C in vitro the levels of metabolites were (mumol/g w.w.): ATP 3.1, PCr 8.5, Cr 5.6, Pi 9.2, LAC 2.1, PYR 0.19, GLU 6.6, ADP 0.44 x 10(-3). The mean steady oxygen uptake of the isolated muscle was 97 nmol O2 x min-1 x g-1 w.w. Thus, the levels of macroergic phosphates and their derivatives are lower after isolation and perfusion of the muscle, but the creatine charge [PCr]/([PCr]+[Cr]) remains stable (0.61 in vivo versus 0.60 in the isolated muscle). This indicates that the steady-state and high energy status of the isolated perfused-superfused gracilis muscle is maintained [corrected].

Phosphocreatine and ATP concentrations increase during flow-stimulated metabolism in a non-contracting muscle.

The gracilis muscle was excised from cold-acclimated rats, placed in vitro, and simultaneously perfused via its artery by high pO2 medium and superfused by low pO2 medium. With a doubling of the perfusion rate (from 50 to 100 microliters/min) phosphocreatine and ATP increased by 39% and 44%, respectively.

Respiration and heat in contracting and non-contracting dissipative muscle.

The oxygen consumption-heat relationship is analyzed using a scheme of reactions underlying muscle aerobic metabolism. The enzymatic chemical reactions of the scheme are considered near equilibrium during a contraction, and far from equilibrium during energy dissipation in a non-contracting state. Implications show that for two different metabolic rates, the proportionality between oxygen consumption and heat is a very specific case, because of restricting necessary conditions.

"Cascade" principle of the control of non-shivering thermogenesis by intravenously infused noradrenaline.

A hypothesis is presented according to which noradrenaline (NA) infused into the blood reaches the biophase of thermogenic cells through three "cascade" steps, namely the blood volume, extravascular space and biophase. The influx rate of the NA entering the system is given by a forcing function and multiplied by cardiac output. Distribution of the NA at each step is identically described by three differential equations.