Derangements in purine nucleotide synthesis and interconversion are associated with a number of clinical disorders. Increased puring biosynthesis leads to overproduction of uric acid, and the resultant hyperuricemia and hyperuricosuria may cause gout and/or renal calculi. Enzymatic defects in the interconversion pathway referred to as the purine nucleotide cycle have been associated with a clinical myopathy. The objective of this proposal is to define the mechanisms which control the rate of purine nucleotide synthesis and hence uric acid production and to better understanding the role of the purine nucleotide cycle in skeletal muscle function.
One specific aim i s to determine what regulates the activity of PP-ribose-P amidotransferase, amidophosphoribosyltransferase, in human cells. The following hypothesis will be tested in these studies: oxygen inactivation of amidophosphoribosyltransferase, which is an iron-sulfur, oxygen-sensitive enzyme, is the initial step leading to degradation of this protein and variability in the sensitivity of this enzyme to oxygen inactivation regulates the activity of this enzyme in the cell. Radio-immunoprecipitation studies are described to define the rate of inactivation and degradation of this enzyme under different growth conditions. The other specific aim is to determine what controls the developmental expression of the purine nucleotide cycle enzymes during myocyte differentiation, what controls flux through this cycle, and what are the physiological consequences of distruption of this cycle. Patients with inherited enzyme defects, i.e. AMP deaminase deficiency, and animal models (rats) of nucleotide cycle disorders will be evaluated for muscle performance, as well as the biochemical basis for muscle dysfunction. Myocyte cell culture models will be used to study the expression of muscle specific isozymes of the purine nucleotide cycle enzymes during differentiation. Intracellular protein turnover and in vitro translation techniques will be used to determine the basis for the increase in activity of the muscle specific isozymes of the purine nucleotide cycle enzymes during differentiation. Studies with purified nucleotide cycle enzymes and contractile proteins will determine if binding of the nucleotide cycle enzymes to contractile proteins plays a role in controlling the activity of these enzymes in the myocyte.
