To mimic what might happen in cells exposed to hypertonicity, the effects of increased osmolarity and ionic strength on cell-free protein synthesis have been examined. Translation of globin mRNA by rabbit reticulocyte lysate decreased by 30—60% when osmolality was increased from 0.35 to 0.53osmol/kg of water by the addition of NaCl, KCl, CH3CO2Na or CH3CO2K. In contrast, equivalent additions of the compatible osmolytes betaine or myo-inositol caused a 40—50% increase in the rate of translation, whereas amino acids (50—135mM) that are transported via system A had no significant effect. Addition of 75mM KCl caused a dramatic fall in the amount of the 43S pre-initiation complex, whereas it was totally preserved when osmolarity was similarly increased by the addition of 150mM betaine. The formation of a non-enzymic initiation complex between rabbit [3H]Phe-tRNA, poly(U) and the 80S ribosomes was unaffected by the addition of 75mM NaCl or KCl, but translation of the complex decreased by 70%. Density-gradient centrifugation of reticulocyte extracts translating endogenous mRNA revealed that addition of 150mM betaine had no effect, whereas addition of 75mM KCl caused a marked decrease in the polysome peak, concomitant with an increase in the proportion of 80S ribosomes and ribosomal subunits, even when elongation was inhibited with fragment A of diphtheria toxin. These results are consistent with the notion that both initiation and elongation are inhibited by unusually high concentrations of inorganic ions, but not by the compatible osmolytes betaine or myo-inositol.
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January 2003
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Research Article|
January 15 2003
Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis
Maurizio BRIGOTTI;
Maurizio BRIGOTTI
∗Dipartimento di Patologia Sperimentale, Università degli Studi di Bologna, 40126 Bologna, Italy
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Pier Giorgio PETRONINI;
Pier Giorgio PETRONINI
†Dipartimento di Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia, Università degli Studi di Parma, 43100 Parma, Italy,
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Domenica CARNICELLI;
Domenica CARNICELLI
∗Dipartimento di Patologia Sperimentale, Università degli Studi di Bologna, 40126 Bologna, Italy
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Roberta R. ALFIERI;
Roberta R. ALFIERI
†Dipartimento di Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia, Università degli Studi di Parma, 43100 Parma, Italy,
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Mara A. BONELLI;
Mara A. BONELLI
†Dipartimento di Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia, Università degli Studi di Parma, 43100 Parma, Italy,
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Angelo F. BORGHETTI;
Angelo F. BORGHETTI
†Dipartimento di Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia, Università degli Studi di Parma, 43100 Parma, Italy,
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Kenneth P. WHEELER
Kenneth P. WHEELER
1
‡School of Biological Sciences, University of Sussex, Brighton BN1 9QG, U.K.
1To whom correspondence should be addressed (e-mail K.P.Wheeler@sussex.ac.uk).
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Publisher: Portland Press Ltd
Received:
July 04 2002
Revision Received:
September 26 2002
Accepted:
October 09 2002
Accepted Manuscript online:
October 09 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 369 (2): 369–374.
Article history
Received:
July 04 2002
Revision Received:
September 26 2002
Accepted:
October 09 2002
Accepted Manuscript online:
October 09 2002
Citation
Maurizio BRIGOTTI, Pier Giorgio PETRONINI, Domenica CARNICELLI, Roberta R. ALFIERI, Mara A. BONELLI, Angelo F. BORGHETTI, Kenneth P. WHEELER; Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis. Biochem J 15 January 2003; 369 (2): 369–374. doi: https://doi.org/10.1042/bj20021056
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