A major, long-term goal of this project is to gain a better understanding of the regulation of folic acid nutrition and metabolism in mammals. We have approached this problem at the molecular level and try to address the physiological significance of our findings with respect to the intact animal. Aside from the obvious relevance to folic acid nutriture in general, this project is particularly relevant to metabolic situations where significant amounts of single carbon units are required such as growth, development and other cases of rapid cell division and growth such as cancer: many cancer chemotherapeutic agents are folic acid antagonists. We have discovered that altered flow through the one-carbon pool brought about by altered vitamin A status and by altered thyroid hormone status is attributable to coordinate changes in two key and irreversible enzymes of the one-carbon pool, 5,10-methyleneTHF reductase (reductase) and 10-formylTHF dehydrogenase (dehydrogenase). We have also discovered that 10-formylTHF hydrolase activity previously attributed to the dehydrogenase is actually a separate mitochondrial enzyme. Studies are proposed to purify and characterize this mitochondrial enzyme and study its role with respect to cytosolic/mitochondrial compartmentalization of folate metabolism. Experiments will also be conducted to quantify the distribution of folates in the mitochondrial and cytosolic compartments as to their polyglutamyl chain length and one- carbon substituent. Additional studies are designed to gain an understanding at the molecular level for the vitamin A and thyroid induced alteration in reductase and dehydrogenase activity and amount. cDNA complementary to corresponding mRNA sequences will be obtained and characterized by standard procedures such as Northern analysis and hybrid select translation. They will then be used as molecular probes in order to characterize the translational versus pretranslational aspects of induction and repression of these enzymes as a function of vitamin A and thyroid hormone status. Relative rates of transcription of the reductase and dehydrogenase genes will be determined as a function of vitamin A depletion and repletion as well as of thyroid status. We will continue our studies with in vivo constant infusion tracer kinetic techniques to quantify flux of carbon through the one-carbon pool to assess the physiological significance of our findings made at the molecular level.
