Analysis of the specific radioactivity of valine isolated from aminoacyl-transfer ribonucleic acid of rat liver

Wallyn, C S; Vidrich, Alda; Airhart, J; Khairallah, Edward A.

doi:10.1042/bj1400545

Cited by 29 publications

(7 citation statements)

References 12 publications

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“…Leucine-specific activities were determined in serum (25), protein-free supemates of the liver (26), and in hepatic leucyl-tRNA at various time intervals. The method used by Wallyn et al (27) for determination of valyltRNA-specific activity was applied to leucyl-tRNA after demonstration that leucine, similarly to valine, originated solely from the amino acyl-tRNA pool. There was no contamination of the leucine bound to tRNA with differently labeled leucine added to the initial homogenates either in the free state or incorporated into serum albumin.…”

Section: Materials L-[u-14c]-(325 Mci/mmol) and L-[n-45-3h]-mentioning

confidence: 99%

Pathogenesis of alcohol-induced accumulation of protein in the liver.

Baraona¹,

Leo²,

Borowsky³

et al. 1977

J. Clin. Invest.

298

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A B S T R A C T Alcohol feeding to rats produced hepatomegaly, associated with enlargement of the hepatocytes. The increase in liver dry weight was accounted for not only by fat but also by protein accumulation, primarily in microsomes and cytosol, with a selective increase in export proteins: concentrations of both immunoreactive albumin and transferrin were augmented in liver microsomes and cytosol of ethanol-fed rats. To investigate the mechanism ofthis protein accumulation, [14C]leucine was injected intravenously and its incorporation into both liver and serum proteins was measured after various time intervals. Rates of synthesis and export were assessed from protein labeling and specific activities of leucyl-tRNA. Synthesis of liver protein and proalbumin were enhanced by chronic ethanol feeding, but this was not associated with a corresponding rise in serum albumin output. Actually, there was a significant retention of the label in liver albumin and transferrin with delayed appearance in the serum of ethanol-fed rats. This indicated that, regardless of the changes in synthesis, the export of protein from the liver into the plasma was impaired. This alteration in export was associated with a decreased amount of polymerized tubulin in the liver of ethanol-treated animals. Thus, both enhanced protein synthesis and defective export contribute to the ethanol-induced accumulation of liver protein, and the decrease in liver microtubules represents a possible site for impairment of protein export.

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Section: Materials L-[u-14c]-(325 Mci/mmol) and L-[n-45-3h]-mentioning

confidence: 99%

Pathogenesis of alcohol-induced accumulation of protein in the liver.

Baraona¹,

Leo²,

Borowsky³

et al. 1977

J. Clin. Invest.

298

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“…The second method involves labeling of cell cultures with radioactive amino acids, extraction of radioactively labeled aminoacyl-tRNA, and measurement of base-sensitive radioactivity either directly by determination of released radioactive amino acid (29,30) or indirectly by determination of counts in putative aminoacyl-tRNA before and after base hydrolysis (27,28). Although used for determination of almost all (except glutamic acid) aminoacyl-tRNAs in mammalian cell cultures and tissues (27,28), only in a few cases (Val, His, Ile) was it confirmed that base-sensitive counts are associated with an amino acid (29,30). Pioneering application of this method for determination of aminoacyl-tRNA in E. coli as early as 1959 was not satisfactory for quantitative purposes due to inadequate methods used for extraction of 14C-labeled aminoacyl-tRNA (16).…”

mentioning

confidence: 99%

Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli

Jakubowski¹,

Goldman²

1984

J Bacteriol

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The cellular content of all 20 aminoacyl-tRNA species was determined in small cultures of Escherichia coli by labeling cells with 3H-amino acids and extraction of 3H-amino acid-labeled nucleic acid by standard procedures. Of 3H-amino acid-labeled material, 25 to 90% was identified as 3H-aminoacyl-tRNA by the following criteria: (i) sensitivity to base hydrolysis with expected kinetics; (ii) association of 3H counts released by base treatment of the 3H-amino acid-labeled nucleic acid with amino acid standards upon paper chromatography of the hydrolysate; and (iii) changes in the amount of 3H-amino acid-labeled nucleic acid recovered from cells as a function of time. Individual aminoacyl-tRNA content was determined with as few as 8 x 107 to 4 x 108 E. coli cells. Although the total number of aminoacyl-tRNA molecules per cell varied only by 10 to 20% among various strains of E. coli, some individual aminoacyl-tRNA families varied two-to threefold among strains. For a given amino acid, the number of aminoacyl-tRNA molecules per cell in E. coli strain K38 growing with a doubling time of 60 min varied from 730 (glutamyl-tRNA) to 7,910 (valyl-tRNA) with a mean value of 3,200. The total number of aminoacyl-tRNA molecules per cell (6.4 x 104) in E. coli K38 was 5.5-fold higher than the number of ribosomes and was equal to 84% of the amount of elongation factor Tu molecules per cell. The ratio of aminoacyl-tRNA to synthetase for 10 amino acids varied from about 1 to 15 with a mean value of 4.7. The turnover of individual aminoacyl-tRNA families in E. coli cells was estimated to be in the range of 1.7 to 8.1 s-1 with a mean value of 3.7 s-1. An estimate of minimum in * Corresponding author. t Visiting Scientist from the Institute

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“…Samples of homogenate were immediately taken into an equal volume of ice-cold 20 % (w/v) trichloroacetic acid and processed as described for plasma. The remaining homogenate was used to isolate aminoacyl-tRNA by a technique previously described (Wallyn et al, 1974).…”

Section: Methodsmentioning

confidence: 99%

“…Two rapid rinse in 0.17M-NaCl were found to be sufficient to eliminate the problem. Controls have already been described which indicated that washing the liver does not alter the specific radioactivity of the tRNA-associated valine (Wallyn et al, 1974) were determined on washed livers.…”

Section: Evaluation Ofmethodsmentioning

confidence: 99%

Compartmentation of free amino acids for protein biosynthesis. Influence of diurnal changes in hepatic amino acid concentrations of the composition of the precursor pool charging aminoacyl-transfer ribonucleic acid

Vidrich¹,

Airhart²,

Bruno³

et al. 1977

Biochemical Journal

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101

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To investigate further the mechanisms by which amino acids are segregated for protein biosynthesis, the distribution of a pulse of [3H]valine was monitored in hepatic amino acid pools at seven intervals in the diurnal cycle of meal-fed rats. Although each condition was characterized by a unique balance between intracellular and extracellular valine, in every case the specific radioactivity of valyl-tRNA at steady state was higher that that of intracellular valine but below the extracellular value. Further, the specific radioactivity of the valyl-tRNA could be accurately predicted if extracellular and intracellular valine were combined in proportions specified by the transmembrane concentration gradient. These observations not only substantiate our earlier conclusions that the amino acids used for protein synthesis do not originate exclusively from either the intracellular or extracellular pools, but also strengthen our theory that the membrane transport system is the physical basis for such compartmentation. On the basis of these data we present a method for measuring the specific radioactivity of the precursor pool for protein biosynthesis in cases where the actual isolation of the aminoacyl-tRNA is not technically feasible, and also suggest a theoretical basis for interpreting the unequal distribution of both total and [3H]valine between intracellular and extracellular fluids.

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Analysis of the specific radioactivity of valine isolated from aminoacyl-transfer ribonucleic acid of rat liver

Cited by 29 publications

References 12 publications

Pathogenesis of alcohol-induced accumulation of protein in the liver.

Pathogenesis of alcohol-induced accumulation of protein in the liver.

Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli

Compartmentation of free amino acids for protein biosynthesis. Influence of diurnal changes in hepatic amino acid concentrations of the composition of the precursor pool charging aminoacyl-transfer ribonucleic acid

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