Abstract
Rat heart mitochondria have been incubated with concentrations of pyruvate from 50 to 500 μ m as substrate in the presence or absence of an optimal concentration of palmitoylcarnitine and with respiration limited by phosphate acceptor. The rate of pyruvate utilization has been determined and compared with the amount of active (dephosphorylated) pyruvate dehydrogenase measured in samples of mitochondria taken throughout the experiments and assayed under V max conditions. At a given pyruvate concentration, differences in pyruvate utilization as a proportion of the content of active pyruvate dehydrogenase are attributed to differences in feed-back inhibition and interpreted in terms of the ratios of mitochondrial NAD + NADH and CoA/acetyl-CoA. Under most conditions, differences in inhibition can be attributed to differences in the CoA/acetyl-CoA ratio. Feed-back inhibition is very pronounced in the presence of palmitoylcarnitine. These parameters are also examined in the presence of dichloroacetate, used to raise the steady-state levels of active pyruvate dehydrogenase in the absence of changing the pyruvate concentration, and in the presence of various ratios of carnitine/acetylcarnitine, which predominantly change the mitochondrial CoA/acetyl-CoA ratio. The latter experiment provides evidence that a decrease in mitochondrial NAD + NADH ratio from 3.5 to 2.2 essentially balances an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in modulating enzyme interconversion, whereas the change in CoA/acetyl-CoA ratio is preponderant in effecting feed-back inhibition. Increasing the free Ca 2+ concentration of incubation media from 10 −7 to 10 −6 m using CaCl 2-ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid buffers is shown to increase the steady-state level of active pyruvate dehydrogenase in intact mitochondria, in the absence of added ionophores. Moreover, this activation is reversible. Effects of Ca 2+ ions are dependent upon the poise of the enzyme interconversion system and were only seen in these experiments in the presence of palmitoylcarnitine.
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