The principal hypotheses to be tested are: 1) changes in hepatic BHMT activity are mediated in BHMT protein levels as a result of increased gene expression and/or increased translation of its mRNA; 2) the pig is a better animal model for nutrition studies on choline oxidation and homocysteine metabolism.
The specific aims are: 1) to determine the molecular mechanisms that mediate low-methionine induced increases in hepatic BHMT activity and to determine if similar mechanisms operate in the kidney; 2) to develop a kinetically competent reaction mechanism and map active-site residues on recombinant human BHMT; 3) to isolate and characterize the human BHMT gene and promoter. To study the expression of hepatic BHMT, rats will be fed methionine-restricted diets designed to produce a range of enzyme activity. After three weeks the rats are sacrificed and livers are removed for analysis of BHMT activity, mRNA and total BHMT protein. Tissue homocysteine, choline, betaine, SAM and SAH will also be determined. Because BHMT has not been detected in rat kidney, a pig model will be used to study dietary-induced changes in kidney BHMT activity. Recombinant human BHMT will be used for detailed kinetic studies to provide information on reaction mechanism. Site-directed mutagenesis will be used to identify active-site residues involved in substrate binding and catalysis. An attempt will be made to crystallize the enzyme for X-ray diffraction studies (in collaboration with Drs. Ludwig and Matthews). Finally the human BHMT gene and promoter will be isolated from a commercial genomic library and characterized with respect to intron/exon junctions, intron sizes and transcription binding elements, hormone response elements and other known regulatory elements.
