THE PHYSIOLOGY AND METABOLISM OF VITAMIN D

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date4/1/797/31/05

ASJC

  • Medicine(all)