Perturbations of rat intestinal brush border membranes induced by Ca2+ and vitamin D3 are detected using steady-state fluorescence polarization and alkaline phosphatase as membrane probes.
Rat intestinal brush border membranes (BBM) were prepared by discontinuous sucrose gradient centrifugation. This specific fraction contained alkaline phosphatase activity. A dramatic decrease in the specific activity of the BBM-bound alkaline phosphatase was observed at different concentrations of Ca2+ and vitamin D3. Studies of the temperature dependence (5-40 degrees) of alkaline phosphatase reveal a change in energy of activation (slope of the Arrhenius plot) at 23.0 +/- 1.1 degrees which was elevated to 27.8 +/- 1.3 degrees in BBM treated with Ca2+, while it was depressed to 17.2 +/- 1.2 degrees in BBM treated with vitamin D3. Membrane lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), was significantly greater in BBM treated with vitamin D3 and significantly lower in Ca2+ treated BBM. A lipid thermotropic transition temperature was observed at 22.2 +/- 1.2 degrees which rose to 28.3 +/- 1.4 degrees in Ca2+ treated BBM and reduced to 17.0 +/- 1.2 degrees in vitamin D3 treated BBM. The biological significance of these results is discussed in terms of modifications in the lipid-protein interactions in BBM, induced by Ca2+ and vitamin D3, and the implications in the physiological intestinal transport processes of calcium.
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D002762:Cholecalciferol / Q000494:pharmacology*; D005454:Fluorescence Polarization; D007422:Intestines / Q000187:drug effects* / Q000648:ultrastructure; D008297:Male; D008560:Membrane Fluidity / Q000187:drug effects; D008871:Microvilli / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D013696:Temperature
D002762:Cholecalciferol; D000469:Alkaline Phosphatase; D002118:Calcium