Precursors For Protein Synthesis Research Articles

Evidence for the lack of subunit exchange between aldolase tetramers in vivo.

The results of a double isotope experiment using 3H- and 14C-labeled leucine as precursors of protein synthesis demonstrated that the aldolase C to A subunit transition which is associated with chick skeletal muscle development involves the preferential synthesis of different aldolase isoenzymes. This developmental system was used to test for subunit exchange between aldolase tetramers in vivo. In a second double isotope experiment, it was found that the 14C:3H ratios of A and C subunits derived from the same heterotetramer were essentially identical, while the isotope ratios of the same subunit type derived from different isoenzymes were considerably different. Had subunit exchange between the isoenzymes occurred, A subunits of a given heterotetramer would have been expected to have higher isotope ratios than the corresponding C subunits. Therefore, these data suggest that subunit exchange between aldolase tetramers does not occur in vivo, at least not in skeletal muscle to an appreciable extent. The results of the present study suggest that all aldolase tetramers are constructed at the time of the initial assembly of newly synthesized subunits, that is, "new" tetramers would not be generated by subunit exchange between already constructed tetramers. In addition, the present work suggests that the degradation of all four subunits of an aldolase tetramer are coupled inasmuch as the subunits would not be reincorporated into other tetramers. Thus, in contrast to some other proteins, it appears that the subunits of the aldolase tetramer turn over coordinately.

Role of N-acetylamino acids in cerebral protein synthesis. I. Incorporation of radioactivity from labelled acetyl and aminoacyl moieties into protein

Incorporation of radioactivity from [ 3H]acetate, [ 14C]aspartate, N-[ 3H-acetyl]aspartate and N-acetyl[ 14C]aspartate into cerebral proteins was investigated in vivo as well as in vitro. Concentrations of both 3H and 14C decreased rapidly in the acid soluble fraction of brain during the period following intracerebral injections of the precursors, [ 3H]acetate and [ 14C]aspartate. Concomitant with this decrease, there was an increase in the incorporation of radioactivity into protein. Only a fraction of radioactivity disappearing from the acid soluble fraction at any given interval of time was transferred to protein. However, the proportion of radioactivity incorporated into protein increased with lapse of time after injection, indicating the formation of progressively larger amounts of metabolites which could serve as more effective precursors of protein synthesis than the original injected compounds. Radioactivities from labelled acetyl and aspartyl moieties of N-acetylaspartate were also incorporated into protein. A comparison of the kinetics of incorporation of 3H and 14C from free acetate and aspartate on the one hand and from N-acetylaspartate on the other suggests that the metabolic pools that inherit acetyl and aspartyl groups from injected N-acetylaspartate are probably different from the ones in which exogenous acetate and aspartate normally enter. [ 14C]Aspartate was incorporated into protein in vitro in a reaction mixture containing cerebral polysomes pretreated with puromycin. A majority of the labelled polypeptides was released into solution under these conditions. Addition of acetate to the reaction medium appeared to stimulate the incorporation of aspartate into both soluble and particulate proteins. [ 3H]Acetate was incorporated mainly into soluble proteins, but this reaction was not dependent on the presence of polysomes and was not affected by added aspartate. The rapid uptake of [ 3H]acetate into soluble proteins and rapid loss of incorporated radioactivity on continued incubation suggest that the acetyl groups were probably added onto completed polypeptide chains in a linkage subject to high turnover. Radioactively labelled aspartate and phenylalanine were incorporated into cerebral polysomes in vitro and the extent of incorporation depended upon the concentration of Mg 2+ ions and the duration of incubation. There was no significant incorporation of N-acetylaspartate under any of the conditions tested. Acetyl-CoA slightly increased the incorporation of both aspartate and phenylalanine in the presence of low Mg 2+ ion concentration and KCl wash proteins, whereas N-acetylaspartate had no effect on this reaction.

Evidence That the Intracellular Pool of Tyrosine Serves as Precursor for Protein Synthesis in Muscle

Several investigators suggested that labeled amino acids are incorporated into protein without first equilibrating with the intracellular pool. Experiments were performed to determine if the intracellular or extracellular [14C]tyrosine represented the precursor for protein synthesis in muscle. Four pieces of rat diaphragm were incubated in the same concentration of [3H]leucine but in different concentrations of [14C]tyrosine. The rates of [3H]leucine incorporation were similar in all the muscles, indicating similar rates of protein synthesis. The 14C counts in protein increased with increasing tyrosine concentrations in the medium, but the moles of tyrosine incorporated into protein were similar only when the counts in protein were divided by the intracellular specific activity. In other experiments muscles were incubated with [3H]leucine and [14C]tyrosine, and then transferred to medium containing [3H]leucine and unlabeled tyrosine. The rate of tyrosine incorporation agreed with leucine incorporation only when calculations used the intracellular tyrosine specific activity. Therefore, the free intracellular tyrosine resembled a precursor for protein synthesis, while the extracellular did not. However, this conclusion did not hold during very short incubations or incubations with low concentrations of tyrosine in the medium, for reasons suggested herein.

Role of the free amino acid pool of the intestine in protein synthesis

1. The accumulation of glycine and its incorporation into protein have been studied in rat intestinal slices in vitro. 2. Pyridoxal HCl, which raises intracellular pool concentration of glycine, similarly increases the rate of glycine incorporation into protein. 3. Kinetic data demonstrates a lag in glycine incorporation into protein, suggesting that the glycine passes through an intracellular pool (or pools) before its incorporation into protein. 4. In vivo experiments using leucine demonstrate that specific activity in the protein is a reflection of the specific activity of the soluble “pool” of leucine. 5. These data are consistent with a model in the intestinal mucosa in which glycine in the extracellular pool is not the direct precursor for protein synthesis, but suggest that glycine and perhaps other amino acids first pass through some intracellular pool(s).

Comparisons between nucleic acids, protein, zinc, and manganese, in rat liver: A relation between zinc and ribonucleic acid.

Extract: The relations of total zinc, manganese, nucleic acids, and protein in the livers of control, hypophysectomized, and calorie-restricted rats were eveluated by regression analysis and analysis of covariance (tables I through V). The most striking findings were the demonstration of a continuous relation between RNA and zinc in the livers of the control rats and of experimental groups (fig. 2), and an absolute increase in the content of manganese in the livers of the calorie-restricted animals (fig. 3). It is suggested that zinc is important for the maintenance of ribonucleic acid synthesis and sub sequent protein synthesis in mammalian tissues. Speculation: In bilogical tissues, ribonucleic acid is a necessary precursor for protein synthesis; possibly zinc, acting as a confactor, is of equal importance.

Transport of 14C incorporated protein in the corticospinal tract of the rat brain.

Abstract— The origin of fibres of a corticospinal pathway in rat brain was located by cortical ablation and Marchi staining and also by electrical stimulation of the motor cortex.Enzyme changes investigated histochemically over 0–14 days post‐injection of 5 μ10.15 m NaCl into the neocortex indicated that very little apparent disturbance of nerve cell metabolism beyond a narrow band adjacent to the path of the microneedle within the cortex had occurred. [14 C]Leucine as a precursor of protein synthesis was used to study incorporation of the amino acid into protein. At the site of injection the maximum level of labelled protein was recorded at 30 min post‐injection, the level decreasing to less than 2 per cent of this at 6 hr.The subsequent axonal flow of labelled protein along the corticospinal pathway was investigated during the period 15 min to 21 days post‐injection. Within 24 hr increasing amounts of labelled proteins were measured caudally, but not more than 6 per cent had migrated beyond 5 mm from the site of injection. At 3 days this percentage had increased to 14.6 per cent, the labelled proteins being distributed in progressively decreasing amounts to a further 13 mm caudal. Very little change from this position was seen during the following 18 days.

14C]Leucine incorporation and axoplasmic flow in the rat brain stem reticular formation

[ 14C]leucine as a precursor of protein synthesis was injected into the reticular formation of adult rats (using a stereotaxic instrument and a glass microneedle) to study incorporation into proteins and subsequent axoplasmic flow. Histochemical observations at the site of injection showed no failure of enzyme activity up to 21 days resulting from the operation. Brain and spinal cords were dissected out at increasing time intervals and [ 14C]leucine-incorporated protein was measured caudally in toto and in the nuclear, mitochondrial, microsomal and supernatant fractions in a liquid scintillation counter to determine axonal migration into the spinal cord. No significant migration of protein was detected. These observations are discussed, and it is suggested that the region investigated consists mainly of short synapsing neurones.