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Regulation of the intestinal absorption of calcium by vitamin D in the rat involves the synthesis or maintenance of specific membrane proteins. A particulate preparation derived from duodenal mucosal homogenates or isolated microvillus membranes contains at least three vitamin D-dependent activities: calcium binding, p-nitrophenylphosphatase, and calcium-dependent ATPase. The membrane calcium-binding activity correlates closely with calcium transport. Solubilization and biochemical purification have led to the separation of the calcium-binding from the enzymatic activities and to the isolation of the calcium-binding protein (CaBP). This membrane protein has an apparent molecular weight of approximately 200,000 in 0.1% Triton X-100 and yields a monomeric subunit of molecular weight 20,500 in sodium dodecyl sulfate. The new protein, named IMCal, is clearly distinguished from the soluble CaBP found in mucosal homogenates and has a higher affinity for calcium than does the soluble protein. The hypothesis is proposed that IMCal is involved in the facilitated entry of calcium into the enterocyte from the lumen of the intestine.

A particulate fraction of rat intestinal mucosal homogenates, termed the "calcium-binding complex," contains three vitamin D-dependent activities: calcium binding of high affinity, calcium-dependent adenosine triphosphatase, and p-nitrophenylphosphatase. These particulate activities vary concordantly with intestinal calcium transport, suggesting that they represent membrane components of the translocation mechanism. The particulate was solubilized with 1-butanol and the activities were resolved partially by gel filtration and by DEAE-cellulose and spheroidal hydroxyl-apatite column chromatography. The Ca-binding activity was separated from the enzymes and isolated as a protein of molecular weight approximately 200,000, as estimated by gel filtration in 0.1% Triton X-100. The membrane protein, named IMCal (intestinal membrane calcium-binding protein), was dissociated with sodium dodecyl sulfate to yield a monomer of molecular weight 20,500 which is clearly distinguishable from the soluble calcium-binding protein (molecular weight 11,500) of rat mucosa. The apparent dissociation constants of Ca2+ of IMCal and of the soluble calcium-binding protein were estimated as 0.37 microM and 2.25 microM, respectively. The vitamin D-dependent activities of the calcium-binding complex are present in isolated intestinal microvillus membranes and may mediate the translocation of calcium from the intestinal lumen to the cytosol.

Prior studies report that plasma membranes of the spontaneously hypertensive rat (Okamoto-Aoki strain) bind less calcium than do the corresponding preparations from Wistar Kyoto controls. The possibility that the differences result from a decrease in the content of integral membrane calcium-binding protein (IMCAL) was explored by the application of immunoassays with polyclonal antisera and a mouse monoclonal antibody. IMCAL binds calcium with relatively high affinity, and its content in many rat tissues is regulated by vitamin D and the level of dietary calcium. Immunoassays of tissue IMCAL demonstrate significant reductions in content in the erythrocyte ghost, intestinal mucosa, kidney, heart, testis, and liver of the spontaneously hypertensive rat as compared to the control strains. The decreases are observed both at 4-5 weeks of age, before the onset of severe hypertension and at 8-9 weeks in the presence of severe hypertension. Moreover, the magnitude of the decrease in erythrocyte IMCAL can account for much of the decrease in the calcium-binding capacity of erythrocyte membranes reported by others. The results are especially significant because an abnormality in the membrane binding of calcium and in the regulation of cytosolic calcium ion concentration could underlie the pathogenesis of the hypertension.

Integral membrane calcium-binding protein (IMCAL) is a vitamin D-dependent integral membrane protein that binds calcium with relatively high affinity (J. Biol. Chem. 225: 10834-10840, 1980). Specific immunoassays for IMCAL utilizing rabbit polyclonal and mouse monoclonal antibodies were developed and applied to studies of its tissue distribution and regulation by vitamin D3 and dietary calcium in the rat. The results indicate that vitamin D-dependent, cross-reactive protein is present in small intestinal mucosa, cecal mucosa, bone, kidney, brain, testis, heart, lung, spleen, and skin. Rats maintained on a low- (0.02%) compared with & high- (2.0%) calcium diet had significantly higher content of IMCAL in duodenal mucosa, cecal mucosa, bone, kidney, brain, testis, and heart. Treatment of rats on the high-calcium diet with 1,25-dihydroxyvitamin D3 increased the IMCAL content of the duodenal mucosa, cecal mucosa, and kidney. The widespread tissue distribution of vitamin D-dependent IMCAL, its close correlation in intestinal mucosa with the calcium transport mechanism, and its occurrence in isolated preparations of enterocyte plasma membranes (microvillus and basolateral membranes) suggest that the protein is involved in the regulation of calcium flux in a number of cell types.