Cell-free translation analysis of the vitamin D-dependent calcium binding protein mRNA activity present in total RNA and polysomal extracts from chick intestine

Abstract

To further elucidate the mechanism(s) involved in the induction of a vitamin D-dependent intestinal calcium binding protein (CaBP), by the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], we have examined by cell-free translation CaBP-mRNA activity present in total cellular RNA extracts or polysomes obtained from chick small intestine. CaBP-mRNA activity was examined by immunochemical analysis of polypeptides completed upon addition of purified intestinal RNA or polysomes to a lysate system. Intestinal polysomes isolated from vitamin D-deficient chicks treated with vitamin D 3, 1,25(OH) 2D 3, or 1α-hydroxyvitamin D 3 were found to complete the synthesis of an immunoprecipitable protein which comigrated with authentic chick intestinal CaBP on SDS polyacrylamide gels. This product was found to represent up to 0.5–1.0% of the total polypeptides directed by polysomes isolated from treated birds but could not be detected among the products directed by intestinal polysomes obtained from vitamin D-deficient birds. A detectable level of polysomal CaBP-mRNA activity was present within 3 h and a maximal activity achieved at 9 h after administration of 1,25(OH) 2D 3. Increases in the levels of cytoplasmic CaBP (measured by radioimmunoassay) were first detected at 6 h, which was significantly later than the polysomal CaBP-mRNA activity. CaBP-mRNA activity in total cellular RNA isolated with guanidinium thiocyanate was also found to be dependent on treatment with vitamin D 3 or 1,25(OH) 2D 3 but represented only 0.1–0.2% of the total mRNA activity. Surprisingly, total RNA and poly(A) RNA were found to direct the synthesis and release of two additional polypeptides that were immunoprecipitated even with an affinity-purified antibody to CaBP. One, an approximately 11,000-dalton polypeptide, was clearly vitamin D dependent; the other, an approximately 38,000-dalton polypeptide, was not. The time course for appearance of CaBP-mRNA activity in total cell RNA extracts following 1,25(OH) 2D 3 treatment was found to be nearly identical to that observed in polysome preparations. These results indicate that induction of CaBP-biosynthesis results, at least in part, from a 1,25(OH) 2D 3-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by 1,25(OH) 2D 3.