Biosynthesis and compartmentalization of rat-intestinal vitamin-D-dependent calcium-binding protein.

We have purified the primary translation product of rat intestinal vitamin-D-dependent calcium-binding protein mRNA from wheat germ and ascites cell-free systems. We show that calcium-binding protein is neither synthesized as a larger percursor nor likely to be exported from the intestinal epithelium. Our conclusions are based on the following observations. (1) The primary translation product, NH2-terminally labeled with formyl[35S]methionine, comigrates with the mature cytoplasmic protein during electrophoresis through denaturing gels. (2) It does not possess a cleavable signal peptide sequence or internal signal equivalent as judged by co- and post-translational cleavage assays in vitro. (3) The NH2 terminus of the cell-free product is acetylated. (4) Comparison of the NH2-terminal amino acid sequences of the primary translation product and cyanogen bromide peptides obtained from the blocked, purified cytoplasmic protein. The kinetics of calcium-binding protein mRNA accumulation and decay in rachitic intestinal epithelium after primary and secondary stimulation with 1,25-dihydroxycholecalciferol (calcitriol) were studied using the cell-free translation system. The results are reminiscent of other steroid-hormone-inducible systems. Both the rate of mRNA accumulation and the peak response were greater after secondary stimulation.