STUDIES ON PURINE BIOSYNTHESIS AND INTERCONVERSION IN THE RAT
BAUGHER, BENNETT WADE
Doctor of Philosophy
A simple, rapid method for the purification to near-homogeneity of rat skeletal muscle adenylosuccinate synthetase was developed. Using enzyme prepared by this method, the molecular weight of the native protein was determined to be approximately 100,000 daltons and several kinetic parameters were evaluated. The kinetic properties of the enzyme indicate that it might be regulated in muscle primarily by the availability of its substrate, inosine-5'-monophosphate. Under some conditions, inhibition by guanosine-5'-diphosphate and by fructose 1,6-diphosphate may also be factors. These properties are consistent with the proposed role of the enzyme in the purine nucleotide cycle in muscle. The activities of the two adenylosuccinate synthetase isozymes in rat liver were measured under varying dietary conditions. The two isoenzymes are separable by ion-exchange chromatography, thus affording a rapid means of analysis. It was found that the expression of the acidic isozyme appears to be coordinated with purine biosynthesis, while the activity of the basic isozyme increases in situations where the animal is metabolizing large amounts of protein. These results indicate that the acidic isozyme is regulated for biosynthesis, while the basic isozyme may be associated with the purine nucleotide cycle. Total activities of adenylosuccinate synthetase and lyase did not change significantly. Since the adenylosuccinate synthetase isozyme content of a tissue might serve as a useful indicator of the direction of metabolism in that tissue, the relative amounts of the two were measured in several different organs. It was found that, under normal conditions, all organs tested contained some of the basic isozyme; it was the only form detectable in skeletal muscle and heart. Liver, kidney, and brain contained both types. In liver the two activities were present in roughly equal amounts, while the acidic form predominates in kidney and brain. The effects of long-term changes in dietary regimen on hepatic adenine and guanine nucleotide pools and de novo purine synthesis and on serum allantoin levels in growing rats were determined. It was found that dietary guanine had relatively little effect on hepatic guanine metabolism, but that adenine is incorporated directly into liver adenine nucleotide pools. High protein and carbohydrate diets also increased the soluble adenine level. The rate of de novo synthesis appears to be unaffected by changes in nucleotide concentration, but is in some way decreased by long-term feeding of large amounts of protein or carbohydrate, indicating that steady-state adaptations to changes in dietary regimen may not follow the same patterns as have been proposed for short-term conditions.