Studies on calcium binding to brush-border membranes from rabbit small intestine

A study was made of the uptake of Ca2+ by brush-border membrane vesicles prepared from rabbit small intestine. The process was found to be time, temperature and substrate concentration dependent, displayed saturability, did not depend on added energy sources and occurred optimally in a pH range of 7.5-8.0. Although the transport of D-glucose by these membrane vesicles responded to changes in osmotic pressure as modified by adding cellobiose to the medium, the uptake of Ca2+ was found not to be osmotically-sensitive. Moreover, the equilibrium uptake value obtained when vesicles were exposed to 0.36 mM Ca2+ was some 60-fold higher than the amount that could have been accommodated by the intravesicular space, calculated from the equilibrium uptake of D-glucose. It was concluded from these results that the uptake involved complete binding of the Ca2+ to the membrane. The ionophore A23187 enhanced the rates of uptake and efflux of Ca2+ without affecting equilibrium values, which suggests that the binding of Ca2+ measured under our conditions was to interior sites of the membrane. The binding capacity was decreased in the presence of 10 mM lidocaine as indicated by a diminution of the equilibrium binding values. Generating an electrochemical potential (negative inside) by addition of valinomycin to vesicles pre-equilibrated with K2SO4, enhanced the rate of uptake of Ca2+. Addition of metal ions, on the other hand, inhibited the uptake, La3+ and Tb3+ being most effective followed by Mn2+, Ba2+ and Mg2+. Na+ and K+ were the least inhibitory. The properties of the Ca2+ uptake process found in rabbit brush-border membranes were compared to those of similar processes occurring in other species.