Binding of calcium to the proteolipid phosphorin

Phosphorin is a phosphate-binding proteolipid isolated from rabbit kidney brush border membrane vesicles that binds inorganic phosphate with high affinity and specificity. This binding of phosphate has a Hill coefficient of 1.92 and an absolute requirement for the presence of a divalent metal. We now describe the binding of Ca2+ to phosphorin that had been depleted of endogenous divalent metal. The dependence of the binding of Ca2+ over the concentration range of 5-100 microM produced a sigmoidal curve, yielding a Hill coefficient of 2.46. (Ruthenium red and La3+ were also potent inhibitors of Ca2+ binding). The divalent metals Mn2+ and Mg2+ were able to inhibit binding of Ca2+ and produced Hill coefficients of 1.75 and 1.98; however, Ba2+ and Sr2+ were less effective in their ability to inhibit binding. In addition, the amine-reactive reagent, 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS), which had previously been found to inhibit binding of phosphate to phosphorin, also completely inhibited binding of Ca2+. We propose that a phosphorin-Me2+ complex forms the molecular species that binds phosphate.