Betaine-homocysteine methyltransferase: Tissue distribution, nutrient regulation, genomic organization, and promoter isolation

Betaine-homocysteine methyltransferase (BHMT) was examined in this thesis. BHMT is an enzyme that catalyzes a methyl transfer from betaine to homocysteine to form dimethylglycine and methionine, respectively. Based on activity assays, and Western and Northern analyses, BHMT was localized to the livers and kidneys of pigs and humans.; In all cases discussed below, the changes in the rat liver BHMT activity mirrored changes in the steady-state mRNA levels. Rats fed the 0.1% methionine diet had BHMT activities that were approximately 4-fold higher (p < 0.05) than the rats fed the 0.3% methionine diet. About 8-fold (p < 0.05) increases were observed when betaine (0.3%), dimethylacetothetin (0.4%) or dimethylpropiothetin (0.4% each) was supplemented to the 0.1% methionine diet. However, cystine supplementation did not affect rat liver BHMT activity. Pair-fed groups showed that rats restricted fed the 0.3% methionine diet had BHMT activity values that were not statistically different or only 70% higher compared to ad libitum rats. In rats fed diets devoid of choline, increasing the concentrations (0.1--0.3%) of methionine in the diet has no effect on BHMT activity. However, in rats fed a 0.1% methionine diet, incremental increases in choline (0--10 mmol) resulted in incremental increases in BHMT activity. This suggests that magnitude of BHMT induction is also dependent on the level of methyl donor in the diet.; The human BHMT gene was cloned. It spanned about 20 kilobases of DNA and contains 8 exons and 7 introns. Using the 5' rapid amplification of cDNA ends technique, the major transcriptional start site was determined to be 26 nucleotides downstream from a putative TATA box.; The electrophoretic mobility (45 kDa) and the nucleotide sequence of the human kidney BHMT has been shown to be identical to liver BHMT. Initial rate data was plotted according to the method of Hanes and the Michaelis Constants were estimated to be 2160 muM and <10 muM for betaine and L-Homocysteine, respectively. These constants were same as the published human liver BHMT constants. Thus, we conclude that kidney and liver BHMT proteins are the product of one human BHMT gene.