This project continues to focus on dihydrofolate reductase, a critical enzyme in the metabolism of the B-vitamin, folic acid. The maintenance of folic acid at the tetrahydro-level by dihydrofolate reductase is critical to cellular survival. Antifolate drugs which target dihydrofolate reductase continue to be widely used in the treatment of cancer, rheumatoid arthritis, and an increasing number of autoimmune diseases. All vertebrate dihydrofolate reductases examined to date have the unique ability to have their catalytic activity significantly stimulated when treated with certain agents known to perturb the tertiary structure of proteins. Chicken liver (ckDHFR) dihydrofolate reductase shows a five-fold to six-fold increase in activity when assayed in the presence of about 5 M urea. Guanidinium (Gdn) salts are among the most interesting protein perturbants because of the strong denaturing activity usually associated with the Gdn ion. However, ckDHFR shows only minimal activation with relatively low concentrations of GdnHC1, i.e., approximately two-fold at 0.2 M. Higher concentrations of GdnHC1 results in rapid denaturation and loss in activity. Since this increase in activity is in the same range as observed in the presence of corresponding concentrations of Na or KC1, it is concluded that this stimulation is due to ionic or salt effects, rather than the well known chaetropic properties of Gdn compounds. However, the Gdn moiety is affecting the protein since equivalent or higher concentrations of salts do not cause similar denaturation despite corresponding activations. On the other hand, the recombinant human DHFR (rhDHFR) does appear to exhibit an activation in response to Gdn HC1. At 0.6 M GdnHC1, the rhDHFR shows about a 40% higher activation than the maximum activation observed with KC1. Similarly, the sheep liver DHFR exhibits a significantly higher activation with 0.65 M GdnHC1 than activation induced by ionic strength. Additional studies with the thiocyanate, acetate, and sulfate Gdn salts revealed similar activations with the thiocyanate and acetate salts. However, GdnSO(4) was found to be a potent inhibitor of all of the DHFRs. Similar results were obtained with NaSO(4). The significance of these results are being interpreted in terms of the activating properties of urea and the known preferential interactions of proteins with the guanidinium ion and salts as well as recent results from x-ray studies on an activated ckDHFR. Studies continue to focus on beta-carotene, its putative antioxidant properties and its relationship to vitamin A. A variety of carotenoids, including beta- carotene, showed no in vivo antioxidant properties when fed to animals deficient in vitamin E and selenium. A copper deficiency had no effect on carotene metabolism in view of the fact that the carotene-converting enzyme, carotene epoxidase, requires copper.

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