Abstract
A recent study reported that protein synthesis was inhibited in rat livers perfused with medium containing vasopressin (Chin, K. -V., Cade, C., Brostrom, M. A., and Brostrom, C. O. (1988) Int. J. Biochem. 20, 1313-1319). The inhibition of protein synthesis caused by vasopressin was associated with a disaggregation of polysomes, suggesting that peptide chain initiation was slowed relative to elongation. In contrast, Redpath and Proud (Redpath, N. T., and Proud, C. G. (1989) Biochem. J. 262, 69-75) recently reported an inhibition of peptide chain elongation by a calcium/calmodulin-dependent mechanism. Therefore, the question remained whether only peptide chain initiation was inhibited or both initiation and elongation were affected by vasopressin. In the present study, vasopressin was found to inhibit protein synthesis in both perfused rat livers and isolated rat hepatocytes. Ribosomal half-transit times in isolated hepatocytes averaged 1.9 +/- 0.1 min with or without vasopressin present in the media, demonstrating that the rate of peptide chain elongation was unaffected by vasopressin. Instead, the inhibition of protein synthesis induced by vasopressin was manifested at the level of peptide chain initiation. Vasopressin treatment resulted in both a 2-fold increase in the number of free ribosomal particles and a greater than 50% decrease in the amount of [35S]methionine bound to 43 S preinitiation complexes. In addition, the activity of eukaryotic initiation factor (eIF) 2B in crude extracts from perfused livers was reduced to 53% of the control value in response to vasopressin. The inhibition of eIF-2B activity was associated with an increase in the proportion of the alpha-subunit of eIF-2 in the phosphorylated form from 9.6% in control livers to 30.7% in livers perfused with medium containing vasopressin. The results demonstrate the novel finding that the inhibition of protein synthesis in vasopressin-treated livers is caused by a reduction in eIF-2B activity due to an increase in phosphorylation of eIF-2 alpha.
Highlights
A recent study reported that protein synthesis was inhibited in rat livers perfused with medium containing vasopressin
The results demonstrate the novel finding that the inhibition of protein synthesis in vasopressin-treated livers is caused by a reduction in eukaryotic initiation factor (eIF)-2B activity due to an increase in phosphorylation of eIF-2cr
We show for the first time that the mechanism of inhibition of protein synthesis in rat liver caused by vasopressin involves phosphorylation of the a-subunit of eukaryotic initiation factor 2 (eIF-2)
Summary
[3H]GDP, and [35S]methionine) were purchased from Amersham Corp., ampholytes The perfusate contained amino acids at 10 times their concentrations in rat arterial plasma [14], except that leucine was present at 5 mM. Hepatocytes were prepared from perfused rat liver by collagenase digestion as previously described [16]. The washed hepatocytes were preincubated for 60 min at 37 “C in buffer containing 2 mM calcium chloride and amino acids at 10 times their concentrations in rat arterial plasma [14] The hepatocytes were incubated (with or without vasopressin) for 20 min prior to measurement of the rate of protein synthesis as described below. Complex Formation-Isolated hepatocytes were incubated as described above except that the concentration of methionine was reduced from 10 to 5 times the concentration found in rat arterial plasma. Twenty min after the addition of vasopressin (5 nM), [35S]methionine (0.2 mCi/ml cell suspension) was
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