An observed requirement for Ca2+ by protein synthesis in mammalian cells will be investigated. The primary goal of the research will involve determining whether the mechanism of this requirement involves direct effects of the cation on components of the protein synthetic apparatus or indirect actions at remote sites which ultimately produce a 3rd messenger modulating the rate of protein synthesis. A subordinate aim entails additional clarification of the potential physiologic significance of Ca2+ as a regulator of protein synthesis and the evaluation of the hypothesis that intracellular free Ca2+ concenrations co-ordinate the rate of protein synthesis with the stimulus-response status of the cell. Initial experiments will involve ascertaining whether Ca2+ affects peptide chain initiation or elongation in the process of mRNA translation by comparing such parameters as polysomal sizes and ribosomal transit times for Ca2+ depleted and Ca2+ restored preparations of intact hepatocytes and C-6 glial tumor cells. Efforts will be made to establish a model cell-free protein synthesizing system in which Ca2+ exerts comparable stimulatory actions to those seen in intact cells. Potential systems to be tested include rabbit reticulocyte lysate and cell-free preparations of hepatocytes and C-6. An observed Ca2+ but not Mg2+ reversible inhbition of reticulocyte lysate protein synthesis by chelators will be examined to ascertain whether the chelator exerts direct inhibitory actions or acts by chelating cation. Gross actions of Ca2+ will be sought on peptide chain initiation and elongation in cell-free systems, with efforts to establish whether the cation acts through a defined intracellular Ca2+-receptor protein such as calmodulin, or by binding directly to one or more proteins associated with the synthetic apparatus. Ca2+ dependent protein phosphorylation-dephosphorylation of such proteins as ribosomal S-6 and elF-2 will be explored. Determination of the physiologic significance of the Ca2+ stimulation of protein synthesis will involve extending an ongoing investigation of the effects of Ca2+ mobilizing hormones on protein synthesis in isolated hepatocytes. This work will include the seeking correlation of changes in free intracellular Ca2+ by Quin 2 fluorescence studies with the rates of overall protein synthesis and serum albumin synthesis and attempts to define the importance of Ca2+ binding to the endoplasmic reticulum in determining the rate of protein synthesis.

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National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Physiological Chemistry Study Section (PC)
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University of Medicine & Dentistry of NJ
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Gmitter, D; Brostrom, C O; Brostrom, M A (1996) Translational suppression by Ca2+ ionophores: reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion. Cell Biol Toxicol 12:101-13
Prostko, C R; Dholakia, J N; Brostrom, M A et al. (1995) Activation of the double-stranded RNA-regulated protein kinase by depletion of endoplasmic reticular calcium stores. J Biol Chem 270:6211-5
Brostrom, M A; Prostko, C R; Gmitter, D et al. (1995) Independent signaling of grp78 gene transcription and phosphorylation of eukaryotic initiator factor 2 alpha by the stressed endoplasmic reticulum. J Biol Chem 270:4127-32
Brostrom, M A; Wong Ling, W L; Gmitter, D et al. (1994) Release of Ca2+ from intracellular organelles by peptide analogues: evidence against involvement of metalloendoproteases in Ca2+ sequestration by the endoplasmic reticulum. Biochem J 304 ( Pt 2):499-507
Wong, W L; Brostrom, M A; Kuznetsov, G et al. (1993) Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells. Biochem J 289 ( Pt 1):71-9
Kuznetsov, G; Brostrom, M A; Brostrom, C O (1993) Role of endoplasmic reticular calcium in oligosaccharide processing of alpha 1-antitrypsin. J Biol Chem 268:2001-8
Prostko, C R; Brostrom, M A; Brostrom, C O (1993) Reversible phosphorylation of eukaryotic initiation factor 2 alpha in response to endoplasmic reticular signaling. Mol Cell Biochem 127-128:255-65
Kuznetsov, G; Brostrom, M A; Brostrom, C O (1992) Demonstration of a calcium requirement for secretory protein processing and export. Differential effects of calcium and dithiothreitol. J Biol Chem 267:3932-9
Prostko, C R; Brostrom, M A; Malara, E M et al. (1992) Phosphorylation of eukaryotic initiation factor (eIF) 2 alpha and inhibition of eIF-2B in GH3 pituitary cells by perturbants of early protein processing that induce GRP78. J Biol Chem 267:16751-4
Rotman, E I; Brostrom, M A; Brostrom, C O (1992) Inhibition of protein synthesis in intact mammalian cells by arachidonic acid. Biochem J 282 ( Pt 2):487-94

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