Bünger R, Mukohara N, Kang Y H, Mallet R T
Department of Physiology, F. E. Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799.
Eur J Biochem. 1991 Dec 18;202(3):913-21. doi: 10.1111/j.1432-1033.1991.tb16451.x.
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].[Pi])) 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]).[Pi]) 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].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol 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 approximately 5-14 mumol 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 approximately 3 to 11 mumol 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 microM to greater than or equal to 16 microM. 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.
在豚鼠心肌中检测了磷酸丙糖异构酶以及甘油醛-3-磷酸脱氢酶/磷酸甘油酸激酶系统的稳态反应物水平。直接测量了包括3-磷酸甘油醛在内的关键糖酵解中间产物，并与肌酸激酶的中间产物进行了比较。在常氧条件下，用5 mM葡萄糖（加胰岛素）灌注非工作状态的Langendorff心脏以及分离的工作心脏，以维持乳酸脱氢酶接近平衡状态。胞质磷酸化电位（[ATP]/（[ADP]·[Pi]））由肌酸激酶得出，游离的[NAD⁺]/（[NADH]·[H⁺]）比值由乳酸脱氢酶得出。在Langendorff心脏中，糖酵解通量从接近零（高钾性心脏停搏）变化到高于正常通量（正常和最大儿茶酚胺刺激）。磷酸丙糖异构酶仅在对照或钾停搏的Langendorff心脏以及缺血后“顿抑”心脏中接近平衡。然而，当糖酵解通量因去甲肾上腺素或生理压力-容积功增加时，该酶就会偏离平衡状态。替代磷酸化比值[ATP]'/（[ADP]·[Pi]））由依赖镁的甘油醛-3-磷酸脱氢酶/磷酸甘油酸激酶系统得出，在生理范围内（0.1 - 2.0 mM）赋予游离镁不同的值。正如所预测的，当高钾停搏使糖酵解几乎停止时（通量约为0.2 μmol C3·min⁻¹·g干质量⁻¹），[ATP]/（[ADP]·[Pi]）和[ATP]'/（[ADP]'·[Pi]'）非常一致。然而，在恢复自发搏动时以及肾上腺素能刺激期间（通量约为5 - 14 μmol C3·min⁻¹·g干质量⁻¹），这两个磷酸化比值之间的相等关系并未消除。相反，当通量因从无工作状态转变为生理压力-容积功而增加时（速率从约3增加到11 μmol C3·min⁻¹·g干质量⁻¹），这两个比值明显不同，表明甘油醛-3-磷酸脱氢酶/磷酸甘油酸激酶处于非平衡状态。仅在肾上腺素能刺激或缺血后心肌“顿抑”期间，而不是由于水力工作负荷本身，3-磷酸甘油醛水平从约4 μM增加到大于或等于16 μM。因此，豚鼠心脏甘油醛-3-磷酸脱氢酶/磷酸甘油酸激酶系统在3-磷酸甘油醛水平升高时（例如由于“顿抑”或肾上腺素能激素），在显著通量下能够实现接近平衡催化的潜力。
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