Calcium binding fraction detected from the brush border membrane during intestinal calcium absorption in the rat

A fraction which had calcium binding activity was detected from the brush border membrane during intestinal calcium absorption in the rat. In situ duodenal loop experiment showed that 45Ca activity in mucosal homogenate, detergent-solubilized whole particulate fraction and detergent-solubilized brush border membrane was maximal at 30 min after the radioactive calcium solution was injected in the loop. The calcium radioactivities did not co-precipitate with anti-IMCal (integral membrane calcium binding protein) antibody. Gel permeation HPLC showed three calcium peaks from both solubilized whole particulate fraction and brush border membrane. In vitro calcium binding assay using the solubilized brush border membrane from the pylorus to proximal ileum showed the existence of specific binding sites with a dissociation constant of 2.19 +/- 0.27 mM (mean +/- SE) while the number of binding sites was 3.4 +/- 0.4 nmol/mg of protein. After in vitro binding, the solubilized brush border membrane was analyzed by gel filtration chromatography. Two calcium peaks, apparently 200 and 1 kDa, were separated and the former peak seemed to be the specific binding fraction but did not contain IMCal. This fraction may bind luminal calcium during intestinal calcium absorption.