RAT VITAMIN-D-DEPENDENT CALCIUM-BINDING PROTEINS - SPECIFICITY OF MESSENGER-RNAS CODING FOR THE 7500-MR PROTEIN FROM DUODENUM AND THE 28000-MR PROTEIN FROM KIDNEY AND CEREBELLUM

Abstract

MRNA extracted from rat duodenum, kidney and cerebellum was translated in a cell-free rabbit reticulocyte lysate system in the presence of L-[35S]methionine. Vitamin-D-dependent Ca-binding proteins (D-CaBP) were identified by immunoprecipitation using antibodies specific to duodenal D-CaBP (7500 MW) and cerebellar D-CaBP (28,000 MW). When duodenal mRNA was translated, the immunoprecipitated polypeptide, obtained using antibodies to duodenal D-CaBP, comigrated with the pure small D-CaBP. Only the addition of unlabeled small duodenal D-CaBP prevented the immunoprecipitation of the major protein. Likewise, when mRNA extracted from the kidney and cerebellum was translated, the product immunoprecipitated by antibodies specific to large mammalian D-CaBP was electrophoretically similar to pure 28,000-MW protein, being displaced only by the addition of unlabeled large D-CaBP. The yield of the duodenal D-CaBP synthesized in the reticulocyte lysate assay was remarkably high (about 10%) compared to that of the large D-CaBP with renal (1%) or cerebellar (0.4%) mRNA. In the absence or presence of microsomal membranes, proteins of similar molecular weight were synthesized, suggesting that the biosynthesis of both large and small D-CaBP do not involve the processing of leader sequences. Duodenal poly(A)-rich RNA was unable to direct the synthesis of large D-CaBP while the mRNAs extracted from kidney and cerebellum did not code for the small D-CaBP. Two distinct mRNAs, coding for small and for large vitamin D-dependent CaBP, are apparently expressed in specific tissues of the rat.