scholarly journals Binding of rat liver and hepatoma polyribosomes to stripped rough endoplasmic reticulum in vitro. Biological or an artifact?

1972 ◽  
Vol 130 (1) ◽  
pp. 19-25 ◽  
Author(s):  
A. A. Hochberg ◽  
F. W. Stratman ◽  
Rainer N. Zahlten ◽  
H. P. Morris ◽  
H. A. Lardy
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Trichloroacetic Acid ◽  
Intact Cell ◽  
Biological Significance ◽  
Thiol Groups ◽  
Inhibition Of Protein Synthesis

Exposed thiol groups do not appear to be related to the binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro. Treating stripped rough endoplasmic reticulum with GSSG did not diminish binding of polyribosomes, suggesting that binding in vitro has no correlation with the inhibition of protein synthesis in vitro reported by Kosower et al. (1971). Thiol reagents, which are known to dissociate ribosomes, did not significantly decrease binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum. Denaturing the protein of 32P-labelled polyribosomes or stripped rough endoplasmic reticulum of liver or hepatoma with heat, trichloroacetic acid, or HClO4 did not alter the binding in vitro. Therefore, the practice of measuring the binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro (Shires et al., 1971b) is an unsuitable indicator of biological significance in the intact cell.

Regulation of protein synthesis by modulation of intracellular calcium in rat liver

1992 ◽  
Vol 263 (5) ◽  
pp. E958-E964 ◽  
Author(s):  
S. R. Kimball ◽  
L. S. Jefferson
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Intracellular Calcium ◽  
Rat Liver ◽  
Fold Increase ◽  
Cytosolic Calcium ◽  
Initiation Factor ◽  
Isolated Cells ◽  
Intact Cells ◽  
Inhibition Of Protein Synthesis

The rate of protein synthesis can be modulated in intact cells by varying the concentration and subcellular distribution of intracellular calcium. Because the biochemical reactions required for the pathway of protein synthesis occur in the cytosol of the cell, it might be expected that protein synthesis would be controlled by free cytosolic calcium rather than the sequestered cation. However, a recent report proposed that maintenance of optimal rates of protein synthesis depends on the amount of calcium sequestered in the endoplasmic reticulum rather than free cytosolic calcium (C.O. Brostrom and M. A. Brostrom, Annu. Rev. Physiol. 52: 577–590, 1990). In the present study, rat livers were perfused with buffer containing various compounds previously shown to alter intracellular calcium concentration and distribution in isolated cells. It was found that conditions designed to cause a rise in free cytosolic calcium had no effect on protein synthesis. In contrast, conditions designed to cause depletion of sequestered calcium resulted in an inhibition of protein synthesis characterized by a reduction in peptide-chain initiation relative to elongation. The inhibition of protein synthesis was further localized to a decrease in the activity of eukaryotic initiation factor (eIF) 2B as measured in extracts from perfused livers. The inhibition of eIF-2B activity was associated with a 2.4-fold increase in the proportion of the alpha-subunit of eIF-2 in the phosphorylated form. In summary, the results of the present study support a model whereby mobilization of calcium sequestered in the endoplasmic reticulum results in an inhibition of protein synthesis in rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Electrophoretic protein blots as aids in choosing fixatives for immunocytochemistry.

1988 ◽  
Vol 36 (4) ◽  
pp. 441-446 ◽  
Author(s):  
J Paiement ◽  
L Roy
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Liver Protein ◽  
Polyacrylamide Gels ◽  
Immunogold Technique ◽  
Immunocytochemical Studies

We used electrophoretic protein blots prepared from polyacrylamide gels to test the effect of different fixatives on the antigenicity of rough endoplasmic reticulum (RER) peptides from rat liver. Protein blots were prepared by the procedure of Towbin et al. (Proc Natl Acad Sci USA 76:4350, 1979), treated with different fixatives, rinsed to inactivate non-specific reactive sites, and then reacted with rabbit polyclonal anti-rat liver RER antibodies, followed by peroxidase-conjugated anti-rabbit antibodies. On the basis of differences in immunostaining densities as determined by densitometry, we found that RER peptides displayed differential sensitivities to various fixatives. Anti-rat liver RER antibodies and the immunogold technique were applied to methacrylate sections of in vitro fixed rat liver rough microsomes. Specific labeling was observed over the microsomes and was shown by quantitation to vary in a similar manner to the immunostaining of specific peptides in protein blots following different fixations. We conclude that protein blots may serve as useful tools for screening the effects of different fixatives on cell antigenicity, and therefore may be helpful in immunocytochemical studies.

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