Our proposed studies on the physiology of 1,25-dihydroxyvitamin D3 are aimed at understanding how the hormone increases calcium (Ca) transport in the intestine. We hypothesize that vitamin D-dependent Ca binding protein (CaBP) plays a vital role in Ca translocation in the enterocyte and that it acts by increasing the activity of the Ca pump at the basolateral membrane, specifically by increasing the activity of the calmodulin sensitive Ca++-Mg++ ATPase. In testing our hypothesis we will examine the structural, metal ion binding and biophysical properties of CaBPs from chicken and human intestine and will examine the influence of the CaBP on the Ca pump and Ca++-Mg++ ATPase in enterocyte basolateral membranes. Metal ion binding to a related 28,000 dalton protein from rat brain and recombinant full length rat brain CaBP and mutants thereof will provide further insights into the nature of these proteins. Finally, we will examine the organization of the gene of a related CaBP from rat intestine.
Specific Aims : 1. (a) We propose to determine the amino acid sequence, Ca and lanthanide binding characteristics and X-ray crystal structure of chick and human intestinal CaBP. (b) Lanthanide and Ca binding studies will be performed on rat brain CaBP, recombinant rat brain CaBP, and mutant rat brain CaBPs. (c) The effect of purified chicken CaBP on the activity of the Ca pump in chicken intestinal basolateral membranes and on purified chick intestinal calmodulin-sensitive Ca++-Mg++ ATPase will be examined. 2) We will isolate a complementary DNA (cDNA) clone for bovine intestinal CaBP from a bovine intestinal cDNA library and isolate and sequence the gene for this protein from a bovine genomic library. Methods: Our methods include the chromatographic isolation of proteins or enzymes, amino acid sequencing by Edman degradation and mass spectrometry, fluorescence and lanthanide binding experiments. Oligonucleotide synthesis, screening of bovine cDNA and genomic libraries, and the nucleotide sequencing of appropriate DNA sequences will be performed. Importance: Information concerning the structure of CaBPs and the mechanism by which they bind Ca and interact with purified Ca translocating enzymes may define how 1,25-(OH)2D3 increases Ca transport and might be important in understanding the pathogenesis of diseases such as osteoporosis or absorptive hypercalciuria in which Ca malabsorption or hyperabsorption occur. An examination of the gene for CaBP would be of importance in understanding how it is organized with respect to certain putative regulatory sequences.
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