Effect of Loading Doses of l-Valine on Relative Contributions of Valine Derived from Protein Degradation and Plasma to the Precursor Pool for Protein Synthesis in Rat Brain

"Flooding" amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and "flooded" (10-28 mM) by comparing the steady-state specific activity of the tRNA-bound valine with that of the plasma valine. Under normal and "flooding" conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; "flooding" with a plasma level of 28 mM raised the brain acid-soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than "flooding" with a plasma level of 17 mM valine, which raised the brain acid-soluble level only to 2.3 mM. The results of these studies show that "flooding" amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.     

Germination of wheat embryos and the transport of amino acids into a protein synthesis precursor pool

Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.

The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate.

     

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.     

Proteome dynamics in complex organisms: using stable isotopes to monitor individual protein turnover rates

The complete definition of changes in a proteome requires information about dynamics and specifically the rate at which the individual proteins are turned over intracellularly. Whilst this can be achieved in single-cell culture using stable isotope precursors, it is more challenging to develop methods for intact animals. In this study, we show how dietary administration of stable isotope-labelled amino acids can obtain information on the relative rates of synthesis and degradation of individual proteins in a proteome. The pattern of stable isotope-labelling in tryptic peptides can be deconstructed to yield a highly reliable measure of the isotope abundance of the precursor pool, a parameter that is often difficult to acquire. We demonstrate this approach using chickens fed a semisynthetic diet containing [(2)H(8)]valine at a calculated relative isotope abundance (RIA) of 0.5. When the labelling pattern of gel-resolved muscle proteins was analyzed, the intracellular precursor isotope abundance was 0.35, consistent with dilution of the amino acid precursor pool with unlabelled amino acids derived from degradation of pre-existing proteins. However, the RIA was stable over an extended labelling window, and permitted calculation of the rates of synthesis and degradation of individual proteins isolated by gel electrophoresis. For the first time, it is feasible to contemplate the analysis of turnover of individual proteins in intact animals.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

Accumulation of free amino acids in growing Xenopus laevis oocytes

The sizes of amino acid pools in growing Xenopus laevis oocytes have been measured. The total free amino acid content per oocyte increases nearly 25-fold during oocyte growth. Together, glutamic acid and aspartic acid account for approximately 59-75% of the total amino acid pool in Xenopus oocytes. On the other hand, methionine and cysteine are the least abundant of the amino acids detected, each accounting for less than 0.7% of the total pool in developing oocytes. It is argued that the acid-extractable amino acid pool represents the precursor pool used in protein synthesis.     

Effect of Depolarizing Agents on the Incorporation of Amino Acids into Soluble Cytoplasmic and Membrane-Bound Proteins of Synaptosome Fractions

Abstract: The incorporation of [U-¹⁴C] protein hydrolysate and [U-¹⁴C]leucine into the trichloroacetic acid (TCA)-insoluble membrane and the soluble synaptoplasm proteins of synaptosomes was studied. Following treatment with the depolarizing agents veratrine, Tityus toxin, or potassium, the specific radioactivity of both precursor pool and proteins was measured to examine the link between protein labeling and the fall in the free amino acid pool due to depolarization-induced release of glutamate and aspartate. By reducing the size of the fall in precursor pool due to depolarization by using a nontransmitter amino acid such as leucine (as compared with the usual use of protein hydrolysate), it was shown that the amount by which the pool is reduced is proportional to the change in the protein labeling observed. These results confirm that membrane depolarization causes a large increase in the labeling of membrane-bound proteins as compared with the soluble synaptosomal proteins.     

ENHANCED CEREBRAL PROTEIN SYNTHESIS IN DEVELOPING HYPOTHYROID RATS: EVIDENCE FOR DELAYED MATURATION

(1) Neonatal hypothyroidism resulted in a 40% increase in the incorporation of [¹⁴C]leucine into protein by cerebral cortical slices from 25-day-old rats. The uptake of the [¹⁴C]-labelled amino acid into the acid-soluble free amino acid pool was similar in hypothyroid and control groups which excluded the possibility that transport differences contributed to the observed differences in incorporation. (2) The conversion of [¹⁴C]leucine in the free amino acid pool to other metabolites was substantially greater in the hypothyroid state compared to euthyroid controls. (3) The correction of the incorporation data for radioactivity associated with [¹⁴C]leucine in the precursor pool, provided an estimate of cerebral protein synthetic rate which was markedly higher in thyroid hormone-deficient-rats compared to litter mate controls. (4) The administration of L-thyroxine to hypothyroid animals for two successive days essentially returned the accelerated metabolism of the precursor pool leucine to normal but failed to ameliorate the increased incorporation into protein. (5) Incubations conducted in the presence of high exogenous leucine levels, to eliminate possible differences in intracellular free amino acid pool size, provided additional evidence for an increased rate of cerebral protein synthesis in 25-day-old hypothyroid rats compared to controls. (6) The results are compatible with a retardation in the normal developmental decline in the rate of cerebral protein synthesis associated with hypothyroidism.     

METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

Biochemical and Autoradiographical Determination of Protein Synthesis in Experimental Brain Tumors of Rats

The rate of leucine incorporation into brain proteins was studied in rats with experimental brain tumors produced by intracerebral transplantation of the glioma clone F98. Incorporation was measured with [14C]leucine using a controlled infusion technique for maintaining constant specific activity of [14C]leucine in plasma, followed by quantitative autoradiography and biochemical tissue analysis. After 45 min the specific activity of free [14C]leucine in plasma was 2.5-3 times higher than in brain and brain tumor, indicating that the precursor pool for protein synthesis was fueled both by exogenous (plasma-derived) and endogenous (proteolysis-derived) amino acids. Endogenous recycling of amino acids amounted to 73% of total free leucine pool in brain tumors and to 60-70% in normal brain. Taking endogenous amino acid recycling into account, leucine incorporation was 78.7 +/- 16.0 nmol/g of tissue/min in brain tumor, and 17.2 +/- 4.2 and 9.7 +/- 3.3 nmol/g/min in normal frontal cortex and striatum, respectively. Leucine incorporation within tumor tissue was markedly heterogeneous, depending on the local pattern of tumor proliferation and necrosis. Our results demonstrate that quantitative measurement of leucine incorporation into brain proteins requires estimation of recycling of amino acids derived from proteolysis and, in consequence, biochemical determination of the free amino acid precursor pool in tissue samples. With the present approach such measurements are possible and provide the quantitative basis for the evaluation of therapeutic interventions.     

Pools and protein synthesis in mammalian cells.

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.     

The effect of starvation on branched-chain 2-oxo acid oxidation in rat muscle.

Oxidative-decarboxylation rates of branched-chain amino acids in rat hemidiaphragm and of branched-chain 2-oxo acids in hemidiaphragm, soleus muscle and heart slices of 110-120 g rats were increased considerably by 3-4 days of starvation, when they were calculated from the specific radioactivity in the medium. When the supply from endogenous protein degradation to the oxidation-precursor pool was severely limited by transaminase inhibitors, oxidative-decarboxylation rates of branched-chain 2-oxo acids rose significantly. Since this apparent increase was relatively larger in preparations from fed rats than from 3-days-starved rats, the differences in oxidation rates with nutritional state became less or even not significant. With rat heart the smaller dilution of the oxidation precursor pool after starvation is in accordance with the reported decrease in protein breakdown. Since protein degradation increases with starvation in skeletal muscles, we suggest that the amino acid pool arising from protein degradation is more segregated from the oxidation precursor pool in muscles from starved than from fed rats. We conclude that starvation increases branched-chain amino acid and 2-oxo acid oxidation in skeletal and cardiac muscle considerably less than has been suggested by previous studies.     

Amino acid analogues: uptake, pool formation and incorporation of phenylalanine and two halogenated derivatives in cultured mammalian cells.

HeLa cells take up Phe and two of its ring halogenated derivatives (pFPhe and pClPhe) with rpaidity, concentrating them against the external medium both at 4 and 37 degrees C. The majority of amino acid (greater than 90%) is accumulated without energy expenditures at 4 degrees C, and can be quickly discharged by normal cell washing procedures in saline. At 37 degrees C the freely-diffusible (FDP) pool is accompanied by another which develops more slowly and cannot diffuse out freely during washings with saline but is extractable with trichloracetic acid (the slowly-diffusible pool, SDP, or more conventionally, the acid-soluble pool). Both of the analogues produced larger pools of the latter type than Phe itself from external concentrations ranging from 10(-5) to 10(-3) M. The incorporation of pFPhe into proteins over these same concentrations ranged from 30 to 90--95% of Phe incorporation, whereas pClPhe showed negligible incorporation. From these and similar analyses it can be concluded that amino acid pools form largely independently of protein synthesis, but bear a close relationship with the external amino acid concentration. The fraction of total uptake into cellular pools entering the SDP was relatively constant over a wide range of external concentrations. pFPhe incorporation into cellular proteins produced the same labelling distribution of Phe. It appears to ener all proteins, the vast majority of which have similar half-lives and turnover rates to Phe proteins. In competition, little or no interference was experienced between the analogue and Phe in uptake and pool formation until excessive amounts of one or the other were present (50--100x). By contrast, incorporation of pFPhe into protein was markedly reduced by the presence of Phe. However, the development of normal or large pools of pFPhe or Phe in cells prior to 3H-Phe incorporation did not affect the linear incorporation pattern of the radioisotope into protein. The relationship of pools to protein synthesis is discussed, and it is concluded that, although the SDP could contain potential precursor molecules for protein synthesis, it does not usually act as the direct supplier of amino acid for protein synthesis. Alternative explanations for precursor supply are discussed.     

Functional significance of the activities of glutaminase and ornithine-ω-aminotransferase in rat brain

The activities of glutaminase, glutamine synthetase (GS), arginase and ornithine amino transferase (orn-T) were studied in three regions of rat brain in heightened neuronal activity by producing convulsions by leptazol. These enzymes were studied in preconvulsive, convulsive and postconvulsive phases. Glutaminase activity was found to increase in all the three regions in the preconvulsive and convulsive phases. GS activity decreased in the preconvulsive phase but rose gradually to the control level when the postconvulsive phase was reached. The activity of arginase decreased in the cerebellum in preconvulsive and convulsive phases. However, in the cerebral cortex there was a decrease in the activity of this enzyme only in the convulsive phase. The results suggest that glutamine acts more likely as a precursor for the neurotransmitter pool of glutamate, while ornithine serves more as a precursor for the neurotransmitter pool of GABA.     

Changes in yeast amino acid pool with respiratory versus fermentative metabolism

The precursors of the amino acid yeast pool are intermediates of either the glycolytic or the tricarboxilic acid pathway (TCA). Accordingly, the influence of the metabolism (fermentative or respiratory) on the internal amino acid pool of the yeast Saccharmyces cerevisiae was established by measuring the intracellular amino acid concentration of the "grande" strain IF1256 and its "petite" mutant either in steady-state or when shifting from fermentative to respiratory conditions. Under steady-state conditions, when the cells only respire, there is a decrease in nearly all the amino acids whose precursors are intermediates of the glycolytic pathway. When the metabolism is exclusively fermentative, the opposite change takes place. This effect is not observed in those amino acids whose precursors come from the TCA cycle. However, in continuous culture and at dilution rates lower than 0.06 h(-1), there is an enormous increase in the concentration of all the amino acids in both strains, whatever their precursor, whereas, in batch cultures, both strains undergo variations in the concentration of most amino acids, when entering stationary growth phase. Results therefore indicate that, the fact that the cells ferment or respire effectively affect their amino acid pool according to their precursors coming from the glycolytic or the TCA pathway, but other parameters, such as growth rate or exponential versus stationary phase, have a much stronger effect on yeast amino acid concentration.     

On the application of compartmental models to radioactive tracer kinetic studies of in vivo protein turnover in animals

A mathematical framework is presented for unifying and extending the various compartmental models and formulae used to calculate fractional protein synthesis and degradation rates in animals from data obtained by infusing labelled amino acids. It is shown how the various schemes can be derived as special cases of the product-precursor model or some three-pool variant. Three-compartment representations, which circumvent the need to measure the specific radioactivity of the precursor pool, are proposed. The mathematical solutions are generally presented in a form that is amenable to parameter estimation by non-linear least squares. The problems of measuring the true precursor pool for protein synthesis are addressed, and theoretical consideration is given to assaying aminoacyl-tRNA.     

Phosphate groups modifying myelin basic proteins are metabolically labile; methyl groups are stable

Young and adult rats received intracranial injections of [33P]orthophosphoric acid. The time course of the appearance and decay of the radioactive label on basic proteins in isolated myelin was followed for 1 mo. Incorporation was maximal by 1 h, followed by a decay phase with a half-life of approximately 2 wk. However, radioactivity in the acid-soluble precursor pool (which always constituted at least half of the total radioactivity) decayed with a similar half-life, suggesting that the true turnover time of basic protein phosphates might be masked by continued exchange with a long-lived radioactive precursor pool. Calculations based on the rate of incorporation were made to more closely determine the true turnover time; it was found that most of the phosphate groups of basic protein turned over in a matter of minutes. Incorporation was independent of the rate of myelin synthesis but was proportional to the amount of myelin present. Experiments in which myelin was subfractionated to yield fractions differing in degree of compaction suggested that even the basic protein phosphate groups of primarily compacted myelin participated in this rapid exchange. Similar studies were carried out on the metabolism of radioactive amino acids incorporated into the peptide backbone of myelin basic proteins. The metabolism of the methyl groups of methylarginines also was monitored using [methyl-3H]methionine as a precursor. In contrast to the basic protein phosphate groups, both the peptide backbone and the modifying methyl groups had a metabolic half-life of months, which cannot be accounted for by reutilization from a pool of soluble precursor. The demonstration that the phosphate groups of myelin basic protein turn over rapidly suggests that, in contrast to the static morphological picture, basic proteins may be readily accessible to cytoplasm in vivo.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Amino Acid Pool Formation in Pseudomonas aeruginosa

The accumulation and behavior of various amino acids in the pool of Pseudomonas aeruginosa (ATCC 9027) were investigated. Patterns of pool formation and maintenance varied with different amino acids tested and were dependent, to a considerable extent, upon the ability of the organism to catabolize the particular amino acid. The establishment of steady-state amino acid pool levels depended upon the activity of the amino acid permease involved and upon the rate of protein synthesis. The presence of a relatively large specific amino acid pool did not affect the formation of a pool of a structurally different amino acid, and a preformed steady-state pool was not displaced by structurally unrelated amino acids. Steady-state amino acid pools decreased rapidly in the presence of inhibitors of energy metabolism and at 0 C. Steady-state internal amino acid pools were found to be in equilibrium with the corresponding external amino acid, present at low levels. A multiplicity of proline pools was demonstrated.     

Compartments of protein metabolism in the developing brain.

We investigated whether the higher rate of amino acid incorporation into immature than into mature brain protein is due to (a) rapid growth, (b) a small rapidly metabolized protein pool, or (c) a higher turnover rate of most of the protein. We measured net growth and the incorporation of [14C]tyrosine or [14C]valine into brain proteins in young rats and mice. The specific activity of the free amino acid pool was kept constant in the tyrosine experiments. Incorporation of tyrosine into protein was continued for up to 30 h by which time the specific activity of protein-bound amino acid reached 1/3 of that of the free (precursor) amino acid. The growth (accretion) of brain proteins was approx. 0.635% per h in mice and rats in the 1-4 day period after birth. In previous studies we found that the turnover rate of the bulk (about 96%) of adult brain proteins is below 0.3% per h. Because of the presence of a small (about 4%) active pool the average turnover rate is 0.6% per h. The present experiments show a degradation rate of 0.7-1.1% per h in the brain proteins of the young. This high metabolic rate is not due to a small rapidly degraded fraction of protein. The very rapid protein fraction previously seen in adult rats is either very small (below 1%) or absent in the young. Thus most of the proteins in the immature brain during the rapid growth phase are formed and broken down at a rate that is approximately three times higher than that of the bulk of proteins in the adult brain. The small active protein pool in the adult on the other hand has a metabolic rate higher than that of the immature brain proteins.