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1. The activity of citrate-cleavage enzyme varies in accordance with the nutritional state of the animal. It is suppressed on starvation and restored on re-feeding after starvation. 2. The increase in enzyme activity that occurs on re-feeding starved animals depends on the diet. It is largest on diets high in carbohydrate and low in fat, and smallest on diets high in fat. Intermediate increases are obtained with balanced diets. 3. The ratio of activities of citrate-cleavage enzyme to acetate thiokinase varies from 2·5 for animals maintained on a balanced diet to 20 for animals re-fed with a diet high in carbohydrate. 4. The changes in activity of citrate-cleavage enzyme correlate with changes in the rate of fatty acid synthesis and provide evidence for the involvement of the citrate-cleavage reaction in fatty acid synthesis.

Kinetic studies with glutamine synthetase showed that the apparent Michaelis constant for glutamine was independent of the concentration of ammonia. Similarly, with other substrate combinations little or no interdependency of apparent Michaelis constants was found. Measurements of the rate of isotope exchange at equilibrium with glutamine synthetase showed that by variation of equilibrium conditions the rate of incorporation of glutamate into glutamine could be made considerably less than, equal, to or considerably greater than the rate of incorporation of inorganic phosphate into ATP. Likewise, with acetate thiokinase, variation of equilibrium conditions resulted in a rate of acetate incorporation into acetyl CoA which was approximately equal to or definitely greater than the rate of AMP incorporation into ATP. This demonstrates that with these enzymes, steps other than covalent bond forming and breaking steps can control equilibrium exchange rates. The results, however, limit mechanistic deductions from exchange data. Glutamine synthetase was found to require prior incubation with ATP for demonstration of maximum initial velocity. The significance of the findings are discussed in relation to alternative hypotheses for the glutamine synthetase and acetate thiokinase reactions, including a new hypothesis based on prior reaction of two substrates before reaction with ATP. Present information appears insufficient to allow a choice among various hypotheses.

1. The activity of citrate-cleavage enzyme declines in alloxan-diabetes. 2. The administration of insulin elevates the activity of the enzyme in livers of normal and diabetic animals. Diets high in glucose or fructose elevate the activity of citrate-cleavage enzyme in normal animals, whereas only the diet high in fructose does so in diabetic animals. These observations parallel the effects of insulin, glucose and fructose on fatty acid synthesis in normal and diabetic animals. The effect of fructose is brought into play more rapidly and is larger than the effect of glucose. 3. With one exception acetate thiokinase shows similar changes at a lower level of activity. 4. The results indicate that insulin acts by increasing glucose utilization, and not by exerting a direct effect on citrate-cleavage enzyme or acetate thiokinase.