Search

It is now established that the mitochondrial production of formate is a major process in the endogenous generation of folate-linked one-carbon groups. We have developed an in vivo approach involving the constant infusion of [(13)C]formate until isotopic steady state is attained to measure the rate of endogenous formate production in rats fed on either a folate-replete or folate-deficient diet. Formate was produced at a rate of 76 μmol·h(-1)·100 g of body weight(-1) in the folate-replete rats, and this was decreased by 44% in folate-deficient rats. This decreased formate production was confirmed in isolated rat liver mitochondria where formate production from serine, the principal precursor of one-carbon groups, was decreased by 85%, although formate production from sarcosine and dimethylglycine (choline metabolites) was significantly increased. We attribute this unexpected result to the demonstrated production of formaldehyde by sarcosine dehydrogenase and dimethylglycine dehydrogenase from their respective substrates in the absence of tetrahydrofolate and subsequent formation of formate by formaldehyde dehydrogenase. Comparison of formate production with the ingestion of dietary formate precursors (serine, glycine, tryptophan, histidine, methionine, and choline) showed that ∼75% of these precursors were converted to formate, indicating that formate is a significant, although underappreciated end product of choline and amino acid oxidation. Ingestion of a high protein diet did not result in increased production of formate, suggesting a regulation of the conversion of these precursors at the mitochondrial level to formate., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The mechanisms which control the production of erythropoietin (Epo) remain enigmatic. Recent data suggest that the half-time of Epo messenger RNA (mRNA) is increased by hypoxia in Hep 3B cells, a human hepatoma line. The post-transcriptional regulation of other rapidly degraded mRNAs is mediated by sequence-specific mRNA binding proteins. In order to determine if Epo mRNA specific binding proteins exist, we probed cytosolic lysates from Hep 3B cells and mouse tissues with radiolabeled Epo RNA. A cytosolic protein that binds specifically to Epo RNA was identified in the Epo-producing, hepatoblastoma Hep 3B cell line by gel mobility shift assay. This protein was identified in both normoxic and hypoxic cells and bound specifically to a 120-base fragment of the 3'-untranslated region (3'-UTR) of Epo mRNA. Binding was completed with unlabeled Epo RNA, but not with granulocyte-macrophage colony-stimulating factor RNA. Ultraviolet light cross-linked Epo RNA-protein complexes migrated as two bands of 70 and 135-140 kD on sodium dodecyl sulfate-polyacrylamide gels. Binding activity was markedly increased in brain and spleen lysates from mice subjected to 24 h of hypoxia. Therefore, the post-transcriptional regulation of Epo expression in response to hypoxia may in part be due to the interaction of Epo RNA with its specific binding protein.