The specific radioactivity of the precursor pool for estimates of the rate of protein synthesis (Short Communication)

Infusion of rats with [U-¹⁴C]glycine resulted in labelling of glycine and serine in tissue proteins. The pattern of labelling in protein more nearly resembled that of the free amino acids in the tissue than in the plasma.     

The asymmetry of the free amino acid pool in sections of the rat brain

The content of free amino acid pool in symmetric regions of cortex, hypothalamus, midbrain and blood of rats which had produced the movement conditioned reflex strengthened by feeding was studied. It was established that the "untrained" rats have higher content of free amino acid pool in their blood. The brain of the experimental animals revealed the biochemical asymmetry which was marked by the differences in free amino acid pool distribution between the left and right halves of the studied regions of brain. It was shown that left sided asymmetry animals i.e. animals with heightened content of free amino acid pool in the left half of the brain dominated in the "untrained" group of rats. The supposition was expressed that this biochemical asymmetry may contribute to the ability of animals to learn.     

Heterogeniety of the amino acid pool for protein synthesis in Ehrlich ascites carcinoma cells

Using a new methodological approach based on a step-wise labelling with [14C] and [3H] amino acids, it was demonstrated that the Ehrlich ascite carcinoma cells are capable of utilizing both intracellular and extracellular amino acid pools for protein synthesis. The inhibition of amino acid transport into the cells is accompanied by a more intensive utilization of the exogenous pool. The described procedure permits to calculate the specific radioactivity of the tRNA-bound amino acid and the absolute rate of protein synthesis.     

Compartmentation of free amino acids for protein synthesis in rat liver

The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [(3)H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2-3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.     

The effect of inhibitors in vivo on protein synthesis and the amino acid pool in the sheep blowfly, Lucilia cuprina.

1. The rates of detoxification of cycloheximide (33 mug/g fresh wt.), puromycin (167 mug/g fresh wt.) and actinomycin D (1 mug/g fresh wt.) were assessed in vivo on the basis of acid-insoluble [14C]leucine incorporation in the sheep blowfly, Lucilla cuprina; these were compared with quantitative estimates which took account not only of incorporation data but also of leucine pool size and turnover. Quantitatively, cycloheximide and puromycin were still at least 50% effective in inhibiting protein synthesis after 6.5 and 24.5h of exposure respectively, whereas values based only on incorporation data suggested that cycloheximide was 83% effective and puromycin completely ineffective after the respective periods. Quantitative estimates also showed that actinomycin D effectiveness increased with increasing exposure over 24.5h, in contrast with values based only on incorporation data, which suggested that it was completely ineffective after 24h.2. All inhibitors affected the dynamic state of the amino acid pool; there was a marked decrease in the rate of leucine-pool turnover as well as an increase in the half-life of leucine in the pool. 3. Inhibition of protein synthesis resulted in changes in leucine-pool size; the most pronounced increase occurred with cycloheximide and puromycin and the most pronounced decreases with actinomycin D. 4. Evidence is presented which suggests that proteolysis is functionally linked to protein synthesis, which determines its rate indirectly.     

A study of the properties of the free amino acid pool and enzyme synthesis in yeast

A study has been made of the distribution and properties of the free amino acid pool in yeast. The depletion of the pool was found to depend upon the energy source used, conditions of growth, and the nature of the exogenous nitrogen source. Pool levels could be restored either by an internal replenishment mechanism or by various nitrogen sources. In the absence of internal replenishment a strong positive correlation was established between the ability of nitrogen compounds to support free glutamic add synthesis and enzyme-synthesizing capacity. Amino acid assimilation by nitrogen-starved yeast was studied and compared with that in other organisms. The significance of these results for the problem of enzyme and protein synthesis in yeast is discussed.     

The effect of insulin on free amino acid pools and protein synthesis in rat skeletal muscle in vitro

1. The effects of insulin in vitro on tissue pools and incorporation into protein of glycine and leucine in the extensor digitorum longus muscle of the rat are reported. 2. It was found that insulin decreased the lag period before the establishment of a linear rate of incorporation of radioactive glycine into protein. 3. The hormone increased the size of the free intracellular glycine pool. No such effect was found for leucine. The accumulation of radioactive glycine in the intracellular fluid compartment was increased. The content of radioactive leucine in the intracellular compartment was decreased. 4. Insulin decreased the specific radioactivity of both glycine and leucine in the extracellular fluid. 5. The hormone also decreased protein catabolism. 6. The effect on protein synthesis was not caused by an increase in the specific radioactivity of the extracellular pool but was possibly related to increased amino acid concentrations in this pool, which could in turn have affected the aggregation of ribosomes.     

ACTH in vivo stimulation of the synthesis of a specific mitochondrial protein in the rat

Protein markers induced by hormones are the necessary probes to study hormone regulation of gene expression. We recently showed that ACTH was able to induce one of these markers in the cytosol of the rat adrenal (Dazord et al, Biochem. J. $\text{176}$, 233–239, 1978). In this paper we described another protein marker whose localization is mitochondrial and whose MW is 134 K. Maximal stimulation is seen 2 hours after ACTH injection. Actinomycin D injected 30 min before or 1 hour after the hormone blocks the stimulation. In hypophysectomized rats both ACTH and cyclic AMP are able to stimulate the synthesis of this protein.     

Drug-induced changes in the composition of the cerebral free amino acid pool

The effects of insulin, hydroxybutyrate, deoxypyridoxine, chlorpromazine, codeine, morphine, puromycin, and cycloheximide on the composition of the free amino acids in mouse and rat brain were tested. Significant changes occurred in a number of amino acids with most compounds tested; the largest was of alanine (a 50% increase with glucose, a 50% decrease with drugs); histidine was often increased, and the nonessential amino acids were mostly decreased. The pattern of changes was somewhat different in the mouse brain from that in the rat brain. Changes of amino acid levels may participate in the pharmacological action of a number of compounds.     

An in vitro amino acid incorporation method for assessing the status of in vivo protein synthesis

A quantitative in vitro amino acid incorporation assay is described which can be used to assess the status of in vivo protein synthesis. The preparation and incubation conditions employed result in constant precursor specific activity and limit amino acid incorporation to completion of nascent peptide chains. Results obtained with this method correlate well with measurements of polyribosome profiles using sucrose gradient centrifugation. The assay is easily applied to a large number of samples, and requires only a fraction of the time and tissue necessary for conventional measures of polysome aggregation. The method has been found suitable for studies of protein synthesis in mouse brain and liver, and in gerbil brain, but not in mouse kidney. Products of in vitro protein synthesis can be separated by standard electrophoretic techniques, allowing a characterization of proteins whose mRNAs are actively translated in vivo.     

The effect of unilateral phrenicectomy on the rate of protein synthesis in rat diaphragm in vivo

The fractional rate of protein synthesis (ks) in the denervated rat-diaphragm has been measured in vivo by the continuous amino acid infusion technique at 1, 3, 5 and 10 days after nerve section, and compared with the rate determined in normal rats. Similar rates of protein synthesis, 14% per day, were found for both the left and right hemidiaphragms in the control animals. In the denervated rats, the rates of protein synthesis in the contralateral control hemidiaphragms were significantly increased as soon as 1 day after nerve section. This is considered to be evidence of a compensatory synthesis in the control tissues. In the denervated hemidiaphragm, the rate of protein synthesis had doubled by the third day after nerve section, but by the fifth day had fallen slightly to a value some 50% greater than that of the controls, and remained at this level for a further 5 days. Based on these measured values of protein synthetic rate, calculated estimates have been made of the rate of protein degradation necessary to account for the reported (Turner, L.V. and Manchester, K.L. (1972) Biochem. J. 128, 789-801) changes in mass of the denervated tissue. During the first three days after nerve section, the rate constant for degradation increased to more than twice the normal rate for skeletal muscle, and remained at this value throughout the peak of the hypertrophy.