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.
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D055598:Chemical Phenomena; D002621:Chemistry; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D064030:S100 Calcium Binding Protein G