The clinical manifestations of abnormal adenosylcobalamin (Ado- Cb1) and methylcobalamin (Me-Cb1) biosynthesis are methylmalonicaciduria and homocystinuria (combined or single) which can produce mental retardation, neuropathy and megaloblastic anemia. The long-term goal of this project is to elucidate the mechanisms of Ado-Cb1 and Me-Cb1 biosynthesis and to gain an understanding of the factors that regulate their production, interconversion and degradation.
The specific aims to be accomplished over the next three years are to: (1) Identify the precursors of Cb1 coenzymes in mammalian cells by high- performance liquid chromatography; (2) Purify Ado-Cb1 coenzyme synthetase from rat liver mitochondria and determine the physical, biochemical and kinetic properties of the complex; 3) Characterize the in vitro conversion of Me-Cb1 to Ado-Cb1 and attempt to purify the enzyme responsible for the initial demethylation reaction; and, 4) Investigate relevant cobala-min- reductant (e.g. thiol groups, reduced flavins) interactions in chemical model systems as a means of understanding potentially significant metabolic steps in the biosynthesis of Cb1 coenzymes. The objectives will be accomplished by: (1) Extracting cultured fibroblasts, leukemia L1210 cells and rat tissues using mild techniques to preserve structural integrity of labile naturally- occurring cobalamins; (2) Characterizing Ado-Cb1 coenzyme synthetase with respect to substrate specificity, Cb1 analog interaction and resolution of the complex into its component parts (Cb1 reduction versus Cb1 adenosylation); (3) Employment of appropriate cellular model systems (e.g. leukemia L1210 cells and Protaminobacter ruber) that are known to have Me-Cb1 demethylating activity; and, (4) Utilization of polarographic, spectrophotometric, and fast-reaction techniques to study corrinoid-ligand interactions.
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