Phosphorylation of parathyroid secretory protein.

Abstract

The phosphorylation ofproteins released into the medium of bovine-parathyroid gland slices or isolated cells incubated with 32Pi has been investigated. The primary protein phosphorylated had a Mr of68,000 and coeluted with newly synthesized parathyroid secretory protein (PSP) on Bio-Gel chromatography and on polyacrylamide gel electrophoresis, Isoelectric focusing of double-labeled samples ([asSimethionine and 32P;) revealed comigration of the two radioactive markers at a pH of.4.6, which was similar to that of purified PSP. Phosphorylation of the Mr 68,000, protein was also-demonstrated in cell homogenates incubated with [7-32P] Methods. Fresh bovine parathyroid glands were obtained from the slaughterhouse. and kept on ice.. They were trimmed of fat and then slices were prepared with a Stadie-'vRiggs tissue slicer. The slices were -incubated with 3$P1 (100 ,ACi/ml) with and without [asS]methionine (10 p.Ci/ml) in a shaking water bath for 2-4 hr at 370C.in phosphate-free Krebs-Ringer bicarbonate-buffer (KRB buffer; 120 mM NaCl/4.75,mM KCl/1.2 mM MgCl2/0.5 or 2.5 mM CaCl2/25 mM NaHCO3, pH 7.4, containing 2 mg ofglucose per ml) in an atmosphere of95% 02/ 5% CO2. At the-end of the incubation period, the medium was separated from tissue by centrifugation at 650 x g for 10 min in -a refrigerated centrifuge. Medium was then centrifuged at 8,500 x g for 20 min and used for further studies. Isolated cells were prepared by the method of Brown et aL (12) and were incubated with 32Pj in phosphate-free KRB buffer. Cells and media were then processed for NaDodSO4/polyacrylamide gel electrophoresis or column chromatography on Bio-Gel A-1.5m (1.2 x 75 cm) in 0.15 M ammonium bicarbonate pH 7.8 buffer.After column chromatography, the radioactive peak fractions were pooled, -lyophilized, and analyzed further.For polyacrylamide gel electrophoresis, the proteins in the medium were precipitated by addition of an equal volume of 20% trichloroacetic acid at 40C. The medium was then washed with 10% and 5% trichloroacetic acid; extracted with ether, and dissolved in NaDodSO4 sample buffer [125 mM Tris-HCl, pH 6.8/1% NaDodSO4/5% (vol/vol) 2-mercaptoethanol/10% (vol/ vol) glycerol]. NaDodSO4/polyacrylamide gel electrophoresis was performed on 10% acrylamide slab gels with the discontinuous buffer system described by Maizel (13). Gel scanning was performed with a densitometer (E. C. Apparatus, St. Petersburg, FL). RESULTS When parathyroid gland slices or isolated cells were incubated for 120 min with 32Piand the medium proteins were precipitated and separated on polyacrylamide gel electrophoresis followed by radioautography, the dominant protein was a 68,000-dalton 32P-labeled protein which eluted in a position corresponding to that of highly purified bovine PSP (Fig. 1). Gel filtration (Bio-Gel A-1.5m) of 32P-labeled medium proteins revealed a peak ofradioactivity in the position where marker 125I-labeled PSP (125I-PSP) eluted (Fig. 2) analyzed by radioimmunoassay for bovine PSP -(5), immunoreactivity appeared only in the dominan...

Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway.

“…6A), the phosphate in all three proteins being predominantly bound to serine residues and to a small extent to threonine, but not to tyrosine, residues (data not shown). The phosphorylation of chromogranin A confirms previous results by others (4,40).…”

Post-translational processing of chromogranin A: differential distribution of phosphorylated variants of pancreastatin and fragments 248–313 and 297–313 in bovine pancreas and ileum

“…CGA is known to be phosphorylated at multiple sites [14][15][16]. In the present study we have identified one of these sites as the region giving rise to the active peptide pancreastatin and another in the sequence immediately C-terminal to pancreastatin.…”

“…We were able to characterize some of these variants as corresponding to the products of two different cDNA sequences which have previously been reported [6][7][8]. It is known that, during biosynthesis, CGA is also modified by phosphorylation, sulphation and glycosylation [2,[14][15][16][17][18]. However, the precise sites at which these modifications occur are not CGA The specificity of antibodies used in the present study is shown: antibody L300 reacts with peptides having an exposed C-terminus corresponding to CGA(306-313); antibody L331 reacts with the amidated C-terminus of bovine pancreastatin.…”

Regulation of protein kinase C by extracellular calcium in bovine parathyroid cells.

“…Phosphorylation of proteins in some systems has been related to hormone secretion or synthesis (25). We previously reported that phosphorylation of parathyroid secretory protein was inversely proportional to calcium concentration (26), suggesting that this protein could be a substrate for protein kinase C. Finally, there is evidence that protein kinase C systems are linked through guanine nucleotide regulatory proteins (G proteins) to membrane receptors (27). In a recent study, Fitzpatrick et al (28) showed that pertussis toxin (which inactivates G proteins) prevents inhibition by calcium of PTH release.…”