STRUCTURE AND FUNCTION OF B12-BINDING PROTEINS

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The objectives are to elucidate the structural and functional properties of cobalamin (Cb1, vitamin B12)-binding proteins which are involved in the cellular uptake and utilization of Cb1, and to apply this information to a number of clinical situations. We will study the functional significance of the observation that Cb1 is preferentially bound by R protein in gastric juice and is not transferred to instrinsic factor until after the R protein moiety is partially degraded by pancreatic enzymes in the small intestine. We will further define the role of intrinsic factor and transcobalamin II in the ileal absorption of Cb1 and study the essentially undefined mechanisms by which Cb1 is released from cells, with particular emphasis on its release from ileal absorptive cells and hepatocytes. We will study the regulation of the two mammalian Cb1-dependent enzymes, L-methylmalonyl-CoA mutase and methionine synthetase, and elucidate the pathways involved in the conversion of Cb1 to its coenzyme forms, Ado-Cb1 and CH3-Cb1. We will study the metabolism of methylmalonic acid and investigate the possibility that it has an important and overlooked biochemical function. We will further define the ileal and neutrophil-hepatocyte mechanisms that prevent the intestinal absorption and tissue dissemination of potentially toxic Cb1 analogues of bacterial origin and determine if these Cb1 analogues are present in blood and tissues in various diseases. We will study the properties and origins of a new class of Cb1 analogues that have recently been observed in human and animal blood and tissues, in multivitamin-mineral pills and animal chows, and in animals exposed to nitrous oxide. We will determine the biologic activities and inhibitory properties of a variety of Cb1 analogues, use them to develop an animal model of Cb1 deficiency, and investigate their activity as chemotherapeutic agents against malignant cells. Methylmalonic acid, homocysteine, and methionine will be assayed in various blood samples using assays based on capillary gas chromatography-mass spectrometry. These assays enable the activities of the two mammalian Cb1-dependent enzymes to be assessed in vivo and will be utilized in studies involving patients with marked and mild Cb1 deficiency, various levels of serum Cb1, various neuropsychiatric diseases, and subjects exposed to nitrous oxide in the work place.