Hormone regulation of cardiac energy metabolism. I. Creatine transport across cell membranes of euthyroid and hyperthyroid rat heart.

Hyperthyroid rat heart was studied with the purpose of identifying the mechanism for the significant decrease in total creatine (free creatine plus phosphocreatine) observed in this pathology and its consequences on heart function. Administration of L-thyroxine in doses of 50-100 micrograms/100 g of body weight during a week resulted in a reversible decrease of the total creatine by 40-50%. Simultaneously, remarkable changes in the creatine transport system across the cardiac cell membranes were observed: both the maximal rate of its active uptake and its passive movement along its concentration gradient were enhanced. In euthyroid hearts, the parameters of creatine uptake (Km approximately or equal to 0.05 mM, Vmax = 20 nmole/min/g dry weight) were similar to those for skeletal muscle and the passive movement of creatine was negligible. In hyperthyroid hearts the latter rate was enhanced to 0.4 mumole min/g dry weight, this showing reversible damages in the cell membrane structure induced by L-thyroxine. This conclusion is consistent with observed penetration of colloidal lanthanum into the cells of hyperthyroid hearts. Perfusion of hyperthyroid rat hearts with 50 mM creatine significantly restored creatine content in the cells, Hyperthyroid hearts with decreased creatine content were found to develop ischemic contracture more rapidly and in higher extent than the euthyroid hearts. Increased sensitivity to ischemic damage may be related to decreased efficiency of energy channeling via phosphocreatine pathway.

[The functional coupling between MM isozyme of creatine phosphokinase (EC 2.7.3.2.) and MgATPase of myofibrils and (Na, K)ATPase of plasma membrane in heart cells]. / Funktsional'noe soprazhenie MM-izofermenta kreatinfosfokinazy s Mg-ATPazoi miofibrill i Na, K-ATPazoi plazmaticheskoi membrany sepdechnykh kletok

The functional role of particulate MM isozyme of creatine phosphokinase (CPK) bound to heart myofibrils has been studied. It has been shown that in the presence of heart myofibrils and MgATP creatine phosphate can be used to rephosphorylate ADP formed in the MgATPase reaction. The rate of creatine phosphate splitting is determined by the kinetic properties of myofibrillar MgATPase and by the kinetic parameters of myofibrillar CPK. It has been found that a purified heart plasma membrane preparation contains high CPK activity. CPK isozyme bound to plasma membrane of heart cells is identical to MM isozyme of CPK and is able to rephosphorylate effectively ADP, formed in the (Na K)ATPase reaction. The rate of creatine phosphate splitting in these coupled reactions is sensitive to ouabain and is determined by the kinetic parameters both of the (Na, K)ATPase and plasma membrane CPK. The results obtained indicate the important role of myofibrillar and plasma membrane CPK in the intracellular energy transport processes.

[Functional characterization of the creatine phosphokinase reactions in heart mitochondria and myofibrils]. / Funktsional'naia kharakteristika kreatinfosfokinaznykh reaktsii, sviazannykh s mitokhondriiami i miofibrillami serdtsa

The kinetic properties of MM-isozyme of creatine phosphokinase (CPK) bound to heart myofibrils have been determined experimentally. It has been shown that CPK isozymes bound to the heart myofibrils and mitochondria are electrophoretically different, but have very similar kinetic properties. For both isozymes the ATP formation reaction is preferable. However, in heart mitochondria the kinetic properties of CPK are compensated for by a tight functional coupling with ATP-ADP translocase. Due to this coupling the ATP formed in the course of oxidative phosphorylation can be used completely for creatine phosphate production in mitochondria. On the other hand, the kinetic properties of myofibrillar CPK isozyme are such that they provide for the effective utilization of creatine phosphate produced in mitochondria for rephosphorylation of AKP formed in the myofibrils during contraction. It is concluded that in the heart cells energy can be transferred from the mitochondria to the myofibrils by creatine phosphate molecules.

[Distribution of creatine phosphokinase isoenzymes (EC 2.7.3.2) in cardiac cells]. / Raspredelenie izofermentov kreatinfosfokinazy (EC 2.7.3.2) v serdechnykh kletkakh

The distribution of the creatinephosphokinase isoenzymes in the cardiac cells was studied by way of fractional extraction combined with an electrophoretic analysis of each of the received fractions. At the same time, the content of the creatinephosphokinase isoenzymes was studied in carefully purified preparations of myofibrils and mitochondria. The results of the conducted analysis indicate a heterogenic distribution of the creatinephosphokinase isoenzymes within the cells: about 30% of the cellular activity of the enzyme is contained in the mitochondria (the mitochondrial isoenzyme) 50% is comprised by the fraction dissolved in the cytoplasm (isoenzymes MM, MB and BB), and about 20%--in the myofibrils (isoenzyme MM), 5% of them being extracted only by a 0.9 M KCl solution. The myofibril preparation freed of the mitochondria also contains some important creatinephosphokinase activity (isoenzyme MM) comparable with their ATP-ase activity. The mitochondrial isoenzymes is found only in these structures of the cell.

[Study of the role of mitochondrial creatine phosphokinase isoenzyme in the process of energy transport in cardiac cells]. / Izuchenie roli mitokhondrial'nogo izofermenta kreatinfosfokinazy v protsesse perenosa energii v serdechnykh kletkakh

The kinetic properties of creatinephosphokinase connected with the mytochondria of the heart were studied. The following kinetic parameters were measured: the Michaelis constants for all substrates -- MgATP, MgADP, creatine and creatinephosphate, and the maximal reaction rates for the direct and reverse reactions catalized by the mytochondrial iso-enzyme of creatinephosphokinase. The peculiarity of this iso-enzyme was demonstrated to consist in the independence of binding adeninenucleotides and guanidine substrates. The kinetic characteristics of this iso-enzyme are such that ATP synthesis from ADP and creatinephosphate is preferable: the Michaelis constant for MgADP is 0.05 mM, for creatinephosphate -- 0.5 mM, for MgATP -- 0.7 mM, for creatine -- 5.0 mM. It was also demonstrated that in the presence of all substrates and products of the reaction the ratio of the speeds of the direct and reverse reactions is regulated by Mg ions: with a low concentration of Mg (below that of ATP) the reaction of creatinephosphate synthesis takes place, with the Mg concentration growing the speed of ATP synthesis increases. Thus, an increase of Mg concentration can, depending on the conditions of the reaction, result in its complete reversal. The regulatory role of Mg is also demonstrated for the case in which ATP is synthesized in the reaction of oxidative phosphorylation, creatinephosphokinase-controlled reaction.

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.