Identification and cell-free translation of mRNA coding for a precursor of parathyroid secretory protein.

J A Majzoub,H M Kronenberg,J T Potts,A Rich,J F Habener

doi:10.1016/s0021-9258(18)50340-9

Abstract

Identification and cell-free translation of mRNA coding for a precursor of parathyroid secretory protein.

Highlights

RNA from bovine parathyroid glands was isolated by density-gradient sedimentation, translated in heterologous cell-free systems, and analyzed electrophoretically on formamide-polyacrylamide gels
A semiquantitative evaluation of the amounts of parathyroid secretory protein-related translation product and Pre-ProPTH biosynthesized by each mRNA fraction was obtained by densitometric scanning of the fluorograms prepared from the electrophoretic gels (Fig. 1)
Parathyroid secretory protein and PTH do not appear to share antigenic determinants as determined by several criteria: our previous studies (l), the immunoprecipitation studies presented in this paper, and the immunocytochemical studies using parathyroid secretory protein and PTH antiserums to determine the subcellular location of these proteins in the parathyroid chief cell

Summary

PROCEDURES

Bovine parathyroid glands were obtained from freshly killed calves at a local abattoir and immediately frozen in liquid nitrogen as described previously [8]. Proteins-Parathyroid secretory protein-related translation products containing 100,000 cpm of [:‘“S]methionine were immunoprecipitated from a reticulocyte cell-free translation mixture and added to [JH]methionine-labeled parathyroid secretory protein obtained from medium in which parathyroid gland slices had been incubated for 4 h and isolated by gel filtration on Bio-Gel P-10 (BioRad, Richmond, Calif.). This mixture was oxidized by treatment with performic acid, and a tryptic digest was prepared as described previously [15]. The efficiencies of the repetitive cleavages of the protein during the degradations were determined by analyses of amino acid yields obtained from an unlabeled myoglobin standard that was codegraded with the radiolabeled protein

RESULTS

O 4 s 12 16 20 24 28 32 36 40 44 48 52 CYCLE NUMBER

DISCUSSION