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

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Abstract
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 precursor 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.