COMBINED GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE PHOSPHOGLYCERATE KINASE IN CATECHOLAMINE-STIMULATED GUINEA-PIG CARDIAC-MUSCLE - COMPARISON WITH MASS-ACTION RATIO OF CREATINE-KINASE

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP]. [P(i)])) was derived from creatine kinase and the free [NAD+]/([NADH]. [H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]'. [P(i)]') was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP]. [P(i)]) and [ATP]'/([ADP]'. [P(i)]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux almost-equal-to 0.2-mu-mol C3. min-1. g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux almost-equal-to 5-14-mu-mol C3 . min-1 . g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from almost-equal-to 3 to 11-mu-mol C3 . min-1 . g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4-mu-M to greater-than-or-equal-to 16-mu-M. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.