The research will characterized the molecular basis of calcium transport across the intestinal mucosa and its regulation by vitamin D. A vitamin D-dependent membrane-associated protein, IMCAL, has been isolated from rat duodenal mucosa. The protein binds calcium with high affinity, and its content in the mucosa correlates closely with the active transport of the cation. The working hypothesis, accordingly, is that IMCAL is a mediator and regulator of the transport. The present investigation will continue the biochemical characterization of the protein, determine its subcellular distribution in intestinal enterocytes and explore its functional role in relation to specific membrane calcium mechanisms. The sequence of the protein will be determined by cDNA cloning and the regulation of IMCAL-specific mRNA studied in intestinal mucosa. Biosynthesis and turnover of the mucosal protein and its regulation by vitamin D will be characterized by examining the incorporation of radioactive amino acids and other precursors. The subcellular distribution of IMCAL in enterocytes will be characterized by isolation of the organelles and immunoassays. The molecular functions of IMCAL will be explored by modulating the tissue content in vivo (vitamin D deficiency versus repletion; low versus high calcium diets), isolating membrane vesicles (microvillus membranes and basolateral membranes) and testing specific membrane functions, including 45Ca uptake, ATP- dependent 45Ca accumulation, calcium-dependent adenosine triphosphatase, and calcium binding. The possibility of intracellular transport of calcium via a specialized membrane vesicle rich in IMCAL will be explored. These studies should contribute to an understanding of a number of human disorders including malabsorption syndromes, metabolic bone disorders and cardiovascular diseases such as essential hypertension.
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