Cellular and luminal forms of rat intestinal calcium-binding protein as studied by counter ion electrophoresis

A novel method, termed counter ion electrophoresis, has been developed to identify calcium-binding proteins. In this procedure labeled calcium (${}^{45}\text{CaCl}{}_2$) is added to the lower (anode) chamber reservoir, and the protein sample is applied to the polyacrylamide gel in the upper (cathode) chamber reservoir. As the calcium migrates toward the cathode and the proteins move toward the anode, calcium-binding sites in the gel become labeled and gel slices, containing these sites, can be identified by liquid scintillimetry after solubilization. Application of this procedure to a study of the vitamin D-dependent calcium-binding protein in growing rats has shown that calcium-binding protein occurs consistently as two protein bands in material from mucosal scrapings, but only as one band (band 1) in material from isolated intestinal cells. Bands 1 and 2 are shown to behave as charge isomers, with band 2 more negatively charged. In weanling rats, material from mucosal scrapings yielded only band 1. When calcium-binding protein from mucosal scrapings of growing rats was prepared in the presence of phenylmethylsufonyl fluoride, the amount of band 2 was reduced. Incubation for 2 h at 37 °C of partially purified calcium-binding protein from mucosal scrapings transformed band 1 to band 2, a conversion inhibited by phenylmethyl-sulfonyl fluoride. Similar treatment of partially purified calcium-binding protein from isolated cells had no effect on band 1. It is concluded that band 1 is the cellular and native form of calcium-binding protein which is transformed enzymatically to band 2 in the presence of luminal material.