Characterization of mutant murine lymphoma cells with altered inosinate dehydrogenase activities.

In order to elucidate the regulatory functions of purine enzymes on the rate of purine biosynthesis, two phenotypically distinct mutant cell lines with altered IMP dehydrogenase activities were isolated from mutagenized cultures of mouse T-lymphoma (S49) cells. A single clone, MYCO-1A, was isolated from wild type S49 cells plated in semisolid agarose containing 1 microM mycophenolic acid. The MYCO-1A cell line was remutagenized, and a clone resistant to 20 microM mycophenolic acid, MYCO-1A-20, was isolated and characterized. Assays of IMP dehydrogenase activity in extracts prepared from mutant cells indicated that the enzyme behaved as a single kinetic species and that the maximal velocity of the mutant enzyme activity is 10-15-fold greater than that obtained from wild type extracts. Altered apparent Km values for substrates and the lack of normal sensitivity to mycophenolic acid of the enzymes from mutant cells imply that the mutants have an alteration in a structural gene coding for IMP dehydrogenase. Measurements of intracellular nucleotides indicated that the mycophenolic acid-resistant clones contained elevated levels of GMP and GTP. Incubation of wild type cells with 1 microM mycophenolic acid caused a depletion of intracellular GTP and GMP levels, an increase in the concentration of IMP, an increase in the total rate of de novo purine synthesis, and a massive excretion of inosine into the culture medium. Similar effects were found for MYCO-1A cells incubated with 5 microM, but not 1 microM, mycophenolic acid. However, neither purine overproduction nor nucleotide pool perturbations were observed for MYCO-1A-20 cells incubated with 25 microM mycophenolic acid. These results suggest that a genetic defect in IMP dehydrogenase activity in humans might lead to excessive purine overproduction with subsequent hyperuricemia and gout.