SUMMARYThymidine kinase (EC 2 . 7 . I .21), an enzyme that catalyses the phosphorylation of thymidine to thymidine-5'-phosphate in the presence of adenosine triphosphate and Mg2+, was not detected in cell-free extracts of Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae or Euglena gracilis. Deoxyribonucleic acid was not specifically labelled in Neurospora crassa grown in the presence of [f4C]thymidine, [14C]thymidine monophosphate or [f4C]thymidine triphosphate. Instead, the isotope was incorporated into both types of nucleic acid in a ratio which approximated the mole ratio of ribonucleic acid to deoxyribonucleic acid. It is considered that the absence of thymidine kinase prevents the specific incorporation of thymidine into deoxyribonucleic acid in intact cells of Neurospora crassa.

The rates of 0, consumption during fatty acid oxidation to acetyl-CoA and to COZ were determined for isolated liver cells from starved rats on the basis of measured rates of respiration and ketogenesis. About 60% of the endogenous 0, uptake was associated with acetyl-CoA formation. The remainder was assumed to represent total combustion of fatty acid to CO, and H,O through the Krebs cycle. In the absence of added fatty acid, I .90 pmol ' inin-' . g-' of acetyl-CoA was generated, of which 1.40pmol . min-' . g-' gave rise to ketone bodies. 0, consumption was stimulated about 30 ::<, by the addition of 2 mM palmitate or 4mM hexanoate. This increase was entirely due to stimulation of 0, consumption related to oxidation of fatty acid to acetyl-CoA. The extra acetyl-CoA produced was channelled into ketone body formation.The gluconeogenic substrate lactate, which increases hepatic demand for ATP, stimulated O2 consumption almost 4076, but in this case the increase was due to an enhancement of Krebs cycle activity, and acetyl-CoA generation was depressed 25 7;. In the absence of added fatty acid, 83 of the total acetyl-CoA produced by cells incubated with lactate was oxidized in the Krebs cycle. Addition of fatty acid caused a further stimulation of 0, uptake which reflected a promotion of acetyl-CoA production with an associated increase in ketone body formation.The uncoupling agent, 2,4-dinitrophenol, stimulated total 0, consumption of cells incubated without exogenous substrate or with added fatty acid. In each circumstance this stimulation of 0, uptake was related entirely to enhancement of Krebs cycle activity and depression of ketogenesis. The large increment in 0, consumption induced by the combination of lactate and 2,4-dinitrophenol in the presence of added fatty acid was likewise attributable solely to a marked stimulation of Krebs cycle flux. Added ethanol caused only a small inhibition of total acetyl-CoA production and had little effect on respiration, but considerably depressed Krebs cycle activity. This depression was relieved by 2.4-dinitrophenol.Oligomycin and antimycin had markedly different effects on fatty acid catabolism although both inhibitors depressed O2 consumption to the same extent. Oligomycin caused only a small diminution of rates of acetyl-CoA formation but induced a strong depression of cellular ATP concentrations and of acetyl-CoA oxidation to C0,. In contrast antimycin had no effect on acetyl-CoA oxidation and brought about only a small decline in cellular ATP levels, but considerably depressed formation of acetyl-CoA.These results are interpreted as indicating that addition of fatty acid to liver cells induces various energydependent processes, including ATP synthesis, reversed electron transfer and metabolite translocation, all of which contribute to the observed stimulation of O2 uptake. It is inferred that oxidation of acetyl-CoA to CO, is preferentially coupled to ATP synthesis whereas oxidation of fatty acid to acetyl-CoA is obligatorily associated with reversed electron transfer....

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The inhibition of glycolysis during ethanol oxidation has been examined in isolated hepatocytes from fasted rats. Glycolytic flux was measured by determining the rate of release of tritium from [6-3H]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are depleted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was restored to normal levels. The inhibition of glycolysis was also partially released by addition of pyruvate and when alcohol dehydrogenase activity was depressed by 4-methylpyrazole. Titrations with this inhibitor revealed inverse linear relationships between the rates of glycolysis and ethanol oxidation. For any given rate of ethanol oxidation, glycolytic flux was lowest and the [lactate]/[pyruvate] ratio highest in the presence of aminooxyacetate, an inhibitor of the malatelaspartate shuttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4-methylpyrazole the inhibition of ethanol oxidation and concomitant restoration of glycolysis were accompanied by a decline in the [lactate]/[pyruvate] ratio, a substantial fall in the rate of reducingequivalent transfer from cytoplasm to mitochondria and an increase in lactate accumulation. These findings imply that the reducing equivalents generated during ethanol oxidation compete with those arising in glycolysis for transfer to the mitochondria. This competition leads to an inhibition of aerobic glycolysis, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD+ that results in depression of anaerobic glycolysis. Allosteric inhibition of 6-phosphofructo-1-kinase due to a decrease in the concentration of fructose 2,6-bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxidation had no effect on glucose phosphorylation as measured with [2-3H]glucose, but induced a substantial increase in cycling between glucose and glucose 6-phosphate.Although the depression of glycolysis associated with ethanol oxidation was reported 20 years ago [l], there remains some uncertainty as to the mechanism of ethanol action [l -41. The prevailing view is that ethanol oxidation brings about an inhibition of glyceraldehyde-3-phosphate dehydrogenase activity, due either to a limitation in the availability of NAD+ or to a direct inhibitory effect of NADH on the enzyme [l, 4, 51. The fall in NAD' and concomitant rise in NADH is considered to be a consequence of the accumulation of reducing equivalents in the cytoplasm as a result of Enzymes. 6-Phosphofructo-1-kinase (EC 2.7