Intracellular Leucine Pool Research Articles

Serendipitous Discovery of Leucine and Methionine Depletion Agents during the Search for Polyamine Transport Inhibitors.

Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies. In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Because S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an inability to biosynthesize spermidine, and spermine. Importantly, we found that this methionine-depletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby obviating the need for a PTI. Using 3H-leucine (the gold standard for LAT-1 transport studies) and JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake of extracellular leucine but instead facilitated the efflux of intracellular leucine pools.

Measurement of Free Intracellular and Transfer RNA Amino Acid Specific Activity and Protein Synthesis in Rat Brain in vivo

Brain protein synthesis was measured in anesthetized adult, male Sprague-Dawley rats by an in situ internal carotid arterial perfusion technique using [3H]leucine. The specific activity of free intracellular leucine and of tRNA leucine were determined by HPLC separation of phenylisothiocyanate (PITC) derivatives of amino acids. The specific activity of the leucyl-tRNA pool rapidly equilibrated with the free intracellular leucine pool within 2 min. The specific activity of the tRNA and free leucine pools in brain reached equilibrium by 10 min. Plasma amino acid specific activity, however, remained threefold higher than the specific activity of tRNA and free leucine pools. Estimates of protein synthesis were 0.62 +/- 0.06 nmol/min/g and were constant between 10 and 30 min of perfusion. The in situ perfusion model for protein synthesis described is a controlled system suited to measurements of protein synthesis in brain that can be applied to the study of brain metabolism under changing physiological conditions.

Effect of Amino Acid Infusion on Relationship between Plasma and Skeletal Muscle Intracellular Leucine Pools

Effect of Amino Acid Infusion on Relationship between Plasma and Skeletal Muscle Intracellular Leucine Pools

Simultaneous response of myocardial contractility and a major proteolytic process to beta-adrenergic-receptor occupancy in the Langendorff isolated perfused rat heart.

The Langendorff isolated rat heart was adapted to the study of minute-to-minute percentage changes in bulk protein degradation by using non-recirculating perfusion. Hearts were perfused at 8 ml/min at 35 degrees C with Krebs-Henseleit buffer containing 11 mM-glucose, and only hearts with regular ventricular rhythm were employed. Proteins were labelled by infusion of [3H]leucine for 0.5 h in vitro. A complete amino acid mixture was then added at 3 times normal rat extracellular concentrations. After labelling, the re-incorporation of [3H]leucine was competitively inhibited by addition of either 4 mM-leucine or 20 microM-cycloheximide. The residual unincorporated radioactivity and the preferentially labelled rapid-turnover proteins were eliminated during a 3 h preliminary perfusion period. The basal rate of release of [3H]leucine and percentage changes were then determined at 1 min intervals, by using each heart as its own control. Leucine metabolism was inconsequential to results. Exchange of intracellular leucine pools with extracellular leucine and subsequent release in effluent perfusate was 95% complete within approx. 2 min. The basal rate of protein degradation was unchanged by electrical stimulation of the heart rate to 360 beats/min or cessation of contractile activity by membrane depolarization under 25 mM-KCl. Infusion of the beta-agonist isoprenaline at 5-500 nM caused a graded inhibition of myocardial protein degradation within 5-6 min, with a maximum inhibition of 30%. This inhibition was sustained for at least 1 h of drug administration and was reversed within 4-6 min of cessation of isoprenaline or simultaneous infusion of 1 microM of the beta-receptor antagonist propranolol. Minute-to-minute adrenergic proteolytic control was a simultaneous co-variable with beta-receptor-mediated inotropic changes in right-intraventricular systolic pressure. Stoppage of the heart in asystole by the Ca2+-channel blocker nifedipine (0.7 microM) delayed the onset, but did not cause sustained reversal, of adrenergic-inhibited degradation, indicating the absence of a direct obligatory mechanistic linkage between the events of the contraction-relaxation cycle and protein degradation in this preparation.

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [ 3H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [ 3H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [ 3H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [ 3H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [ 3H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [ 3H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.

Protein synthesis in pulmonary alveolar macrophages Source of amino acids for leucyl-tRNA

Extracellular, intracellular and tRNA-bound leucine pools of the adherent pulmonary alveolar macrophage were examined to determine the relationships between them and the precursor for protein synthesis. When cells were cultured in media of various leucine concentrations, the patterns of isotope distribution in intraceullar and extracellular leucine did not correlate with the patterns seen in protein-bound leucine. Hence, the free leucine pools cannot be used reliably as precursors for calculating rates of protein synthesis. tRNA-bound leucine, however, behaved isotopically as if it were the precursor. Constant synthetic rates were calculated using the tRNA specific activity over a wide range of leucine concentrations. In addition, by measuring the tRNA-bound specific activities of three different amino acids, leucine, valine and phenylalanine, and their respective specific activities in protein, we were able to calculate independently three separate but identical synthetic rates. At physiological amino acid concentrations, the macrophage intracellular leucine pool and the tRNA-bound leucine pool received less than half of their amino acids from extracellular sources. At 5 mM external leucine, the intracellular specific activity was indistringuishable from that of the medium leucine, but the specific activity of the tRNA-bound leucine pool remained only about 50% that of the extracellular value. The most straightforward interpretation of why the tRNA-bound leucine did not flood with external label under conditions where the intracellular pool has reached equilibrium is to propose that some portion of the leucine for protein synthesis is derived directly from protein turnover before the degradation products have mixed with the common amino acid pool.

Enhanced biosynthesis of human skin collagenase in fibroblast cultures from recessive dystrophic epidermolysis bullosa.

Using a sensitive, specific immunoprecipitation method, the biosynthesis of human skin collagenase was studied in fibroblast cultures from patients with recessive dystrophic epidermolysis bullosa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of solubilized immunoprecipitates showed two 3H-labeled procollagenase species that comigrated with those harvested from control cultures. Recessive dystrophic epidermolysis bullosa cultures accumulated increased amounts of collagenase. Both the initial rate of accumulation of intracellular enzyme and the rate of secretion were enhanced, suggesting that excessive accumulation is related to increased synthesis. Because the turnover of labeled collagenase was unaltered, the accumulation could not be attributed to diminishing enzyme degradation. No preferential incorporation of [3H]leucine into recessive dystrophic epidermolysis bullosa collagenase occurred. Furthermore, the mutant cultures displayed no alteration in total protein synthesis, the intracellular leucine pool, or the growth kinetics of the cells. Cells from a patient with dominant epidermolysis bullosa did not show enhanced accumulation of collagenase. The levels of collagenase synthesized in vitro correlated with those observed previously in vivo in recessive dystrophic epidermolysis bullosa patients, suggesting that this biochemical trait is pathogenetically significant in the disorder.

Isolation and characterization of revertants of the mammalian temperature sensitive leucyl‐tRNA synthetase mutant tsHI

Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5 degrees C.

Washing Macrophage Suspensions Inhibits Protein Synthesis

SummaryWhen suspensions of rabbit alveolar macrophages are washed repeatedly at 4° by centrifuging and resuspending the cells, incorporation of [14C]- or [3H]leucine into protein is inhibited, the inhibition increasing with increasing number of washings. Although washing decreases the protein content of macrophages, it does not change the DNA content or the phagocytosis of 3H-labeled E. coli. Washing results in a depression of net leucine transport and a reduction in the translational activity of the polyribosomes, but it does not appear to change the intracellular leucine pool. Mac-rophage monolayers which are washed do not show inhibition of protein synthesis.

Protein metabolism in preimplanted mouse ova

Intracellular specific radioactivity of an amino acid (leucine) was measured in preimplantation mouse ova. The measured specific activity along with leucine incorporation rates allow calculation of the protein synthetic rate in mouse ova. Unfertilized ova, fertilized ova, two-cell ova, and blastocysts convert amino acid to protein at the rate of 8.60, 7.36, 6.92, and 56.68 × 10 −13 moles of amino acid per hour per ovum, respectively. The specific activity measurements also allowed the calculation of intracellular leucine pool size. For the unfertilized, fertilized, two-cell, and blastocyst stage, the endogenous leucine pool was 11.9, 15.9, 6.4, and 62.4 × 10 −14 moles of leucine, respectively. Most of the increase in pool size in the blastocyst probably results from the increase in total volume of cells. Protein degradation measurements indicated a marked difference between protein turnover in the two-cell and blastocyst stage. Approximately 10% of the protein at the two-cell stage turns over with a half-life of 18.3 hr and 35% of the protein at the blastocyst stage turns over with a half-life of 11.2 hr. The large remaining fraction of protein turns over much more slowly.

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding. III. Protein synthesis in vitro in the submandibular and parotid gland after stimulation of secretion in vivo.

After stimulation of the protein secretion by pilocarpine or feeding the rate of incorporation of [3H]-leucine increases in the acinar cells of the parotid gland of the rat while the secretory cells of the submandibular gland show a moderate decrease (Kuijper et al., 1975b). Since the rate of labelled amino acid incorporation depends on the specific radioactivity of the amino acid used, which is not easy to determine in vivo, experiments in vitro were performed to get an idea of the influence of this factor on the measured changes in [3H]-leucine incorporation. In vitro both cell types showed a more pronounced but essentially identical reaction as in vivo. Since in these experiments the specific radioactivity of the extracellular leucine is the same whether fragments of stimulated or unstimulated glands incorporate the radioactive amino acid, the increase of incorporation in the parotid and the decrease in the submandibular cells cannot be ascribed to differences in specific radioactivity of leucine, unless the intracellular leucine pool should show great differences between secreting and non-secreting cells. However, in vitro the submandibular gland cells under both conditions appear to use the extracellular leucine for their protein synthesis (or a small compartmentalized pool in rapid exchange with the extracellular pool). In the parotid cells the whole intracellular pool showed such a rapid exchange with the extracellular one that for practical reasons one may say that these cells, too, rely on the extracellular specific radioactivity of leucine in their protein synthesis. We conclude that the rat parotid gland cells show a rapid and substantial increase of protein synthesis after stimulation of their enzyme secretion, while the submandibular gland cells do not.

Gastrointestinal hormones and function of pancreatic islets: studies on insulin secretion, 3H-leucine incorporation and intracellular free leucine pool in isolated pancreatic mouse islets.

Secretion and biosynthesis of (pro-) insulin were studied in isolated pancreatic mouse islets in the presence of secretin, pancreozymincholecystokinin, gastrin and glucagon, at 0, 100 and 300 mg⁄100 ml glucose. Incorporation of aHleucine into the proinsulin and insulin fraction obtained after gel nitration of the islet proteins on a Sephadex G 50 fine column was taken as parameter of insulin synthesis. Specific radioactivity of free intracellular leucine pool in the isolated islets was determined under different conditions. In the absence of glucose, pancreozymin was the only substance to stimulate insulin secretion.When glucose was present in the medium, glucagon was able to potentiate insulin output. Secretin and gastrin did not change insulin release. Insulin synthesis, found to be stimulated by glucose like insulin secretion, was unaffected by all 3 gastrointestinal hormones whether or not glucose was present. (Pancreatic) glucagon, on the other hand, potentiated glucose-induced insulin synthesis. Spe...

Secretion and protein metabolism in the rat pancreas in vitro

1. 1. Carbamylcholine (> 1 · 10 −6M), pancreozymin (> 0.1 unit/ml) or caerulein (> 2 ng/ml) inhibit the incorporation of L[4,5- 3H 2]leucine intoacid-insoluble proteins of the rat pancreas in vitro. Incorporation of other amino acids, L-[1 3H 2]-methionene and L-[3,5- 3H 2]tyrosine, is also inhibited. 2. 2. The incorporation of labeled leucine into proteins proceeds linearly for at least 2 h in the presence of 0.8 mM leucine but slows down in the absence of the carrier amino acid (26 μM leucine). In both cases, the incorporation is decreased by carbamylcholine immediately after 5 min incubation. 3. 3. During incubation in “leucine-free” (26 μM) medium, the intracellular leucine pool remains constant and its specific activity regularly but rapidly declines. 0.1 mM carbamylcholine increases both the pool size and its specific activity to a maximum after 30 min of incubation. In a medium containing leucine (800 μM), 0.1 mM carbamylcholine also increases the intracellular leucine pool as well as its specific activity. 4. 4. Glutamic acid, aspartic acid and alanine incorporated label from [1- 14C]-leucine; the initial slope of their specific activity curve during the incubation is steeper for the carbamylcholine-treated gland. 5. 5. The following conclusions are made: at low secretion rate there is independence of protein synthesis and the extrusion-secretion process. At high secretion rate there is: (a) an inhibition of protein synthesis at a later step than the availability of the precursor amino acid, and (b) an increase of leucine catabolism.

Microtechnique for Determining the Specific Activity of Radioactive Intracellular Leucine and Applications to in Vivo Studies of Protein Synthesis

Abstract A microtechnique has been developed for the determination of specific activities of radioactive leucine in the nanogram range, with an accuracy of at least 7%. The technique depends on the coupling of [3H]leucine to [14C]2,4-dinitrofluorobenzene. By use of inulin in the incubation medium, a correction can be made for extracellular leucine contaminating the tissue, and thus the specific activity of the intracellular leucine pool can be estimated. The technique has been applied to determining rates of protein synthesis in a protein-secreting gland, the galea of the silkmoth, Antheraea polyphemus, after progressive exposure to actinomycin D. Actinomycin D is shown to superinduce incorporation of [3H]leucine in the differentiation-specific product of the galea, the zymogen of cocoonase. However, superinduction is illusory; when changes in specific activity of the intracellular pool are taken into account, no actinomycin effect on zymogen synthesis is apparent.

Studies in vitro on Free Amino Acid Pools and Protein Synthesis in Rat Jejunum

1. The uptake and incorporation in vitro of radioactive leucine in rat jejunum has been studied. — 2. The total free intracellular leucine pool makes only a minor contribution to tissue protein synthesis. — 3. Amino acid is incorporated into protein more readily from the mucosal than from the serosal side of gut sacs. — 4. The results of these experiments can be interpreted to show that in rat jejunum in vitro leucine is incorporated into protein without prior equilibration with the total free intracellular pool.