The objectives of this research proposal are to elucidate the structural and functional properties of cobalamin (Cbl, vitamin B12)-binding proteins which are involved in the cellular uptake and utilization of Cbl, and to apply this information to a number of clinical situations. We will study the functional significance of the observation that Cbl is bound by R protein in gastric juice and is not transferred to intrinsic factor until after the R protein moiety is partially degraded by pancreatic enzymes in the small intestine, and will evaluate further a new dual label Schilling test for pancreatic exocrine function that is based on this observation. The recently isolated cell surface receptors for intrinsic factor and transcobalamin II will be used to study the mechanisms by which cells take up Cbl and the results will be compared with similar studies involving the cellular uptake of iron, which will utilize the recently isolated cell surface receptor for transferrin. We will explore the feasibility of developing an automated fluorescent method for counting reticulocytes using anti-transcobalamin II receptor antibodies. We will study the synthesis of Cbl coenzymes, develop radioimmunoassays for the two human Cbl-dependent enzymes methylmalonyl-CoA mutase and methionine synthetase, investigate their regulation, and use this information to define and develop new methods of treatment for patients with inborn errors of Cbl metabolism. Cbl analogues of bacterial origin, Cbl analogues recently observed in human plasma and animal tissues, and the deleterious effects on Cbl metabolism caused by nitrous oxide, will be studied to determine if they are a cause of disease in man.
