Though there is extensive information on the requirements, metabolism, and coenzymic functions of B-complex vitamins, there is a paucity of knowledge concerning the means by which such essential nutrients are transported in mammalian cells. Most earlier studies have focused on gastrointestinal uptake, circulatory transport, and the ultimate gross disposition in target cells. Little attention has been given to the specific mechanisms involved in uptake (and efflux) of several B vitamins from the diverse mammalian cell types. Our laboratory has acquired general information on the different means for uptake of certain B vitamins by hepatocytes isolated from rat liver and by renal proximal tubular cells from rat kidney. These studies reveal that the entry of riboflavin and vitamin B6 in both liver and kidney cells is facilitated and exhibits specificity; hence, plasma-membrane carrier systems were indicated. Recent work by others using fluorescence technique has identified flavin-binding protein(s) in the hepatocyte plasma membrane. Preliminary work in our laboratory using affinity chromatography has led to the isolation of B6-binding proteins from the luminal plasma membranes of renal cells. The present proposal is to elucidate the specific mechanisms by which riboflavin and vitamin B, enter and exit both liver and kidney cells. The proteins responsible for facilitating transport through the plasma membranes of such cells will be isolated by rupture of viable cells, density-gradient centrifugation to differentially select plasma membranes, solubilization of protein components, and chromatographic selection of those that have greatest affinity for the vitamin. The binding proteins will be characterized for molecular properties (M.W., K(a) for vitamin, specificity, etc.) and reconstituted into vesicles to see to what extent transport is enhanced. Antibodies will be made and in some cases fluorescence-labeled to verify function and localization of the vitamin-binding proteins within the plasma-membranes, vesicles, and cells. The proposed research will significantly expand understanding of how riboflavin and vitamin B6 enter and exit liver and kidney cells differentially involved in metabolic conversion and excretion. Moreover, potential differences in vitamin transport among normal and abnormal cells, either vitamin-deficient or tumor, will provide critical knowledge concerning nutrient utilization in these pathological states.
