Studies of a mutation affecting pyrimidine degradation in inbred mice.

SUMMARY Dagg, Coleman, and Fraser (Genetics, 49, 979 (1964)) first described a mutation in C57B1/6 mice that resulted in a 3-fold decrease in the capacity of the intact animal, or liver homogenates, to degrade uracil to ,8-alanine, COZ, and am- monia when compared to Rf/J mice. We have examined the mechanism for this metabolic defect by assaying sepa- rately the three enzymes involved in pyrimidine degradation in mouse liver in mouse strain Rf/up, from which Rf/J was derived. The three enzymes, dihydrouracil dehydrogenase, dihydropyrimidinase, and fi-ureidopropionase, are readily separable by diethylaminoethylcellulose chromatography, indicating that the metabolic defect is not the result of a mutation in a single protein with multiple enzymatic activi- ties. Assays of each of the three enzymes separately indi- cate that only the last enzyme of the pathway, P-ureidopro- pionase, is defective in the C57B1/6 strain. This activity in C57B1/6 is 16% of the activity of the Rf/up strain. The mutation most likely affects the structure of the enzyme, since enzyme partially purified from C57B1/6 is more thermo- labile, and has a decreased affinity for substrate than enzyme from Rf/up mice. The P-ureidopropionase has a hereto- fore unrecognized requirement for sulfhydryl reduction for maximal activity. The results indicate that the activity of this enzyme is rate- limiting for the over-all degradation of pyrimidines to /3- alanine in liver of both mouse strains. Several recent studies from this laboratory have explored the use of inbred mouse strains as a source of mutations for studying enzyme regulation in higher organisms. In one study, Ganschow and Schimke (1) showed that independent mutations among closely related C57Bl strains affected the catalytic activity and rate constant of degradation of hepatic catalase. In another study, Doyle and Schimke (2) showed that a mutat,ion first studied by Coleman (3) affected the amount of &aminolevulinate dehydratase by regulating the rate of specific enzyme synthesis without affecting the rate constant of degradation. These studies, then, indicate that inbred mouse strains can provide a * This research was supported by Grant P-427 from the Amer- ican Cancer Society and by Grant GM-14931 from the United States Public Health Service. wealth of material for the study regulation specific enzyme metabolism in mammals. One of the most interesting and potentially important muta- tions in inbred mouse strains is that described in 1964 by Dagg, Coleman, and Fraser (4) which involves the degradation of pyrimidines by the three enzyme pathway depicted in Fig. 1, a pathway studied in a number of laboratories (5-8). Dagg et al. (4) found that Rf/J mice were capable of converting injected 14C-2-uracil to 14C02 at a rate three times that of C57Bl/6 mice and determined that this capacity was regulated by two alleles at a single locus with codominant inheritance properties. Using substrates labeled with 1% in position C-2 of uracil, dihydro- uracil, and the ureido carbon of ureidopropionic acid, and deter- mining evolution of 14C02 in crude liver extracts, they concluded that all three enzymes in the pathway were diminished 3- to 8-fold in the C57B1/6 relative to Rf/J. Such a mutation is particularly important, since it would represent a single muta- tion affecting all enzymes of a given pathway, and as such would represent evidence based on mutants of an operon in animal tissues. Because of its potential importance we have under- taken a more extensive study of the biochemical basis of the mutation. We have developed sensitive assays which assay each enzyme separately, rather than depend on each subsequent enzyme in the pathway (see Fig. 1) as did Dagg et al. (4). We have found that only the last enzyme of the pathway, fi-ureido- propionase, is affected by the mutation. It is most likely that the mutation affects the structure of the enzyme, since the en- zyme from the C5761/6 mice is more thermolabile, and has a higher apparent