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Tetrahymenawere grown with shaking for 17 hours in the presence and absence of AMP. Under these conditions, glycogen content increased but there was a marked inhibition of glyoxylate oxidation and of the incorporation of label from acetate into glycogen during a 1-hour incubation. AMP treatment also caused and increase in the rate of incorporation of [3H]uracil into RNA which was maximal about 2 hours after the start of growth. Experiments were performed in which cells from log and transition phase cultures were incubated with acetate, pyruvate, and octanoate, but with only one substrate labeled in any one flask. Measurements were made of the amount of label incorporated from each labeled substrate into glycogen, lipids, CO2, and glutamate, and the results were analyzed according to a previously established model for the structure of metabolism in Tetrahymena. It was found that the model, which utilizes three pools of acetyl-CoA, permitted a quantitative fit to the data. Analysis of the data showed that AMP treatment reduced the rate of the Krebs cycle, the rate of glyconeogenesis, and the rate of pyruvate utilization in both log and transition phase cells from these labeled substrates. Incorporation of label from acetate into glutamate, however, was increased by AMP treatment in log cells and decreased in transition phase cells. AMP treatment markedly decreased the fraction of label entering into glyconeogenesis from the peroxisomal pool of acetyl-CoA. There was no change in the ATP:ADP ratio in log cells treated with AMP, and a slight decrease in the sum ATP + ADP. In transition phase cells, however, the ATP:-ADP ratio decreased and the sum of ATP + ADP also increased compared to control cells. Since many of the changes in intracellular flux patterns occurred in both log and transitional cells, changes in enzyme levels or control mechanisms other than the ATP:ADP ratios must be invoked to explain the effects of AMP on metabolism.

Experiments were done in which control cells and cells exposed to tolbutamide for 17 hours were incubated for 1 hour with labeled pyruvate, acetate, and octanoate, and the amount of label appearing in CO2, glycogen, lipids, and glutamate was measured. In separate experiments ATP and ADP levels were also measured in control and tolbutamide-treated cells. The results of the multiple label experiments were interpreted in accordance with a previously developed model of the metabolism of Tetrahymenawhich has three pools of acetyl-CoA. It was found that in log phase cells there were significant increases in octanoate oxidation in the peroxisomal and inner mitochondrial pools of acetyl-CoA, and a shift of acetate utilization away from the peroxisome and into the inner mitochondrial compartment. Incorporation of label from [1-14C]acetate into glutamate was reduced by tolbutamide. Carbon flow from the peroxisomes for glyconeogenesis was markedly reduced, but carbon flow for glyconeogenesis from the outer mitochondrial pool was increased. In transition phase cells, however, carbon flow toward glyconeogenesis was reduced from both the peroxisomes and the mitochondria. The cell content of ATP was increased by exposure of the cells to tolbutamide; the increase was more pronounced in log phase cells. It is suggested that tolbutamide does not uncouple oxidative phosphorylation in Tetrahymenabut instead alters the intracellular pattern of carbon flow because of an effect on the rate of flow of substrates across the inner mitochondrial membrane.

The compartmentalization of acetyl-CoA metabolism in Tetrahymenahas been studied by measuring the incorporation of label from [1-14C]acetate, [2-14C]pyruvate, and [1-14C]-octanoate into CO2, lipids, glycogen, and glutamate in experiments in which these substrates were used singly, in pairs, and all together. The results of these studies require that there be at least three pools of acetyl-CoA in Tetrahymena, two in the mitochondria and one in the peroxisomes. A steady state model was developed which allows a quantitative analysis of the metabolic fate of acetyl-CoA derived from acetate, pyruvate, and octanoate. The model shows that pyruvate enters a different mitochondrial pool of acetyl-CoA than acetate or octanoate, which accounts for the relatively high proportion of [2-14C]pyruvate label incorporated into glycogen. About three-fourths of the acetate utilization occurs in the mitochondria, the remainder via the peroxisomal pool of acetyl-CoA for lipogenesis. Only part of the octanoate utilization is accounted for by β oxidation in the peroxisomes; a pathway is also required allowing the incorporation of label from octanoate into lipids without prior oxidation to acetyl-CoA. Labeled acetyl-CoA used for glyconeogenesis comes from both the peroxisomal and the outer mitochondrial pools of acetyl-CoA, more of it coming from the former. The experiments were performed on well aerated cultures in both the logarithmic and transitional phases of growth. Differences in metabolic flux patterns between cells at these two stages of growth are small; lipogenesis was decreased and glutamate output and pyruvate utilization were increased in transition as compared to log cultures.