The long term objectives of this project are to characterize precisely the interaction of cobalamin with macromolecules required for intestinal absorption, transport, and cellular uptake of this vitamin and to develop more readily applicable radioassays by which to identify, measure, and study the metabolism of the physiologic and non-physiologic cobalamins. Toward these objectives, we have the following specific aims: a) To characterize the mechanism by which the intrinsic factor (IF)-cobalamin complex binds to the IF-receptor protein and facilitates the internalization of cobalamin. For this we will use radioimmunoassay and immunocytochemical analysis of ileal epithelial cells. b) To identify and characterize immunoreactive forms of the cobalamin binding proteins fractionated by gel-filtration and ion exchange chromatography using specific RIA to identify these proteins. c) To develop radioimmunoassay techniques to measure the specific physiologic forms of cobalamin and use these analytical methods to measure the concentration of these coenzymes directly in blood and tissue extracts. d) To isolate and characterize the nonphysiologic forms of corrinoid compounds in plasma using chromatographic separation and ligand binding radioassays. e) To develop radioimmunoenzymatic assays to study the synthesis of the two physiologic coenzymes, adenosyl cobalamin and methyl cobalamin and to characterize the enzymes required for these biosynthetic processes. The information obtained and the techniques developed in this project would extend our understanding of the disorders of cobalamin absorption and transport which may occur with drugs, toxins and liver disease; and the analytical techniques to measure cobalamin coenzymes could provide a far simpler method for determining acquired and inherited cellular disorders of cobalamin coenzyme synthesis.
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