scholarly journals Prolyl-tRNA-based rates of protein and collagen synthesis in human lung fibroblasts

1981 ◽  
Vol 198 (2) ◽  
pp. 249-258 ◽  
Author(s):  
J N Hildebran ◽  
J Airhart ◽  
W S Stirewalt ◽  
R B Low
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Amino Acid ◽  
Collagen Synthesis ◽  
Human Lung ◽  
Specific Radioactivity ◽  
Cell Protein ◽  
Free Proline ◽  
Wide Range ◽  
The Relationship

Knowledge of the dynamics of collagen turnover requires information regarding rates of synthesis of this group of connective-tissue proteins. The relationship of various amino acid pools to the tRNA precursor pool used for protein synthesis is known to vary between different cell types and tissues, even for essential amino acids. We studied extracellular, intracellular and tRNA-proline pools in cultured human lung IMR-90 fibroblasts to determine the relationship between them as candidate proline precursor pools for total protein and collagen synthesis. Time-course experiments showed that the three proline pools attained distinctly different steady-state specific radioactivities (extracellular greater than intracellular greater than tRNA) at the extracellular proline concentration of 0.2 mM. The kinetics of radioisotope incorporation into cell protein and collagenase-digestible protein indicated that the intracellular free proline pool could not be used reliably as a precursor for calculating synthetic rates. However, tRNA-proline behaved isotopically as if it were the precursor and provided synthesis rates 2-3-fold higher than those calculated by using either free proline pool. The incorporation of labelled lysine and leucine was constant over a wide range of extracellular proline concentrations. Fractional rates of protein synthesis based on tRNA-amino acid were the same with [3H]phenylalanine as with [3H]proline. The specific radioactivity of cell-associated hydroxyproline reached a steady-state value 8-10h after radioisotope administration which matched the mean tRNA-proline specific radioactivity, suggesting that tRNA-proline is not isotopically compartmentalized. A model of cellular proline-pool relationship is presented and discussed.

scholarly journals The relationship between placental protein synthesis and transfer of amino acids

1983 ◽  
Vol 210 (1) ◽  
pp. 99-105 ◽  
Author(s):  
M J Carroll ◽  
M Young
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Guinea Pig ◽  
Specific Radioactivity ◽  
Protein Amino Acid ◽  
Naturally Occurring ◽  
Placental Protein ◽  
The Relationship ◽  
Radioactivity Measurements

The relationship between placental protein synthesis and transfer of amino acids from mother to foetus was studied in the guinea pig, by using [U-14C]-lysine, -leucine, -glycine, -aspartate and -alpha-aminoisobutyrate. The uptake of label by protein was 12-16% of total label transferred. Cycloheximide inhibited incorporation of all naturally occurring amino acids into protein by 81-96% and transfer by 62-75%; the concentration of label in the free pool was increased for each. These findings were confirmed when specific-radioactivity measurements were made with L-[U-14C]lysine. The transfer of the non-protein amino acid alpha-aminoisobutyrate was not significantly decreased by cycloheximide. A model, linking protein synthesis to the generation of a transfer pool of amino acids, is proposed whereby inhibition of protein synthesis decreases the amount of amino acid available for transfer.

scholarly journals A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine

1980 ◽  
Vol 192 (2) ◽  
pp. 719-723 ◽  
Author(s):  
P J Garlick ◽  
M A McNurlan ◽  
V R Preedy
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Large Dose ◽  
Accurate Determination ◽  
Specific Radioactivity ◽  
Rapid Rise ◽  
Rapid Procedure ◽  
Wide Range ◽  
Convenient Technique

A rapid procedure for measuring the specific radioactivity of phenylalanine in tissues was developed. This facilitates the accurate determination of rates of protein synthesis in a wide range of tissues by injection of 150 mumol of L-[4-(3)H]phenylalanine/100 g body wt. The large dose of amino acid results in a rapid rise in specific radioactivity of free phenylalanine in tissues to values close to that in plasma, followed by a slow but linear fall. This enables the rate of protein synthesis to be calculated from measurements of the specific radioactivity of free and protein-bound phenylalanine in tissues during a 10 min period after injection of radioisotope.

scholarly journals Measurement of protein synthesis in rat lungs perfused in situ

1980 ◽  
Vol 188 (1) ◽  
pp. 269-278 ◽  
Author(s):  
Clyde A. Watkins ◽  
D. Eugene Rannels
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Free Amino ◽  
Plasma Concentrations ◽  
Specific Radioactivity ◽  
Sole Source ◽  
Bicarbonate Buffer ◽  
Normal Plasma ◽  
Rat Lungs

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-14C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O2/CO2 (19:1) or O2/N2/CO2 (4:15:1)]. [U-14C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [14C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [14C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.

scholarly journals Metabolism of rabbit skin collagen. Differences in the apparent turnover rates of type-I- and type-III-collagen precursors determined by constant intravenous infusion of labelled amino acids

1979 ◽  
Vol 181 (1) ◽  
pp. 75-82 ◽  
Author(s):  
S P Robins
Keyword(s):  
Amino Acid ◽  
Collagen Synthesis ◽  
Specific Radioactivity ◽  
Deae Cellulose ◽  
Type Iii Collagen ◽  
Type I ◽  
Turnover Rates ◽  
Type Iii ◽  
Type I Procollagen ◽  
Skin Collagen

Growing rabbits were infused for up to 10 h with labelled proline, tyrosine and leucine to achieve plateau conditions within body free pools, for [3H]proline infusion, blood free-proline specific radioactivity remained constant after about 1 h. For individual animals, type-I- and type-III-collagen precursors were isolated by precipitation with (NH4)2SO4 and DEAE-cellulose chromatography. Experiments where 3H- and 14C-labelled proline and tyrosine were infused concurrently for different periods of time showed that type I procollagen reached plateau specific radioactivity within 3 h and 90% of the plateau value after 2 h infusion, corresponding to a calculated apparent t 1/2 of less than 26 min. Plateau values for type I procollagen were taken as precursor amino acid pool specific radioactivities. The type-III-collagen-precursor fractions consistently showed lower rates of label incorporation and, by assuming that both type I and type III collagens are synthesized from the same amino acid pools, kinetic analysis revealed an apparent t 1/2 for the isolated type-III-collagen precursors of 3.9 h. For proline, there were large variations between animals in the ratio between the precursor pool for collagen synthesis and the skin homogenate free pool (0.31 +/- 0.13, mean +/- S.D.), so that collagen-synthesis rates based solely on total tissue free-pool values for proline are subject to large and inconsistent errors.

scholarly journals The relationship between citrulline accumulation and salt tolerance during the vegetative growth of melon (Cucumis melo L.)

2009 ◽  
Vol 55 (No. 2) ◽  
pp. 51-57 ◽  
Author(s):  
H.Y. Dasgan ◽  
S. Kusvuran ◽  
K. Abak ◽  
L. Leport ◽  
F. Larher ◽  
...  
Keyword(s):  
Amino Acid ◽  
Salt Tolerance ◽  
Cucumis Melo ◽  
Citrullus Lanatus ◽  
Molar Ratio ◽  
Free Proline ◽  
Nacl Concentration ◽  
Induced Changes ◽  
The Relationship ◽  
Final Amount

Citrulline has been recently shown to behave as a novel compatible solute in the <i>Citrullus lanatus</i> (<i>Cucurbitaceae</i>) growing under desert conditions. In the present study we have investigated some aspects of the relationship which might occur in leaves of melon seedlings, also known to produce citrulline, between the capacity to accumulate this ureido amino acid and salt tolerance. With this end in view, salt-induced changes at the citrulline level have been compared in two melon genotypes exhibiting contrasted abilities to withstand the damaging effects of high salinity. Progressive salinization of the growing solution occurred at 23 days after sowing. The final 250 mmol/l external NaCl concentration was reached within 5 days and further maintained for 16 days. In response to this treatment, it was found that the citrulline amount increased in fully expanded leaves of both genotypes according to different kinetics. The salt tolerant genotype Midyat was induced to accumulate citrulline 4 days before the salt sensitive Yuva and as a consequence the final amount of this amino acid was twice higher in the former than in the latter. Compared with citrulline, the free proline level was found to be relatively low and the changes induced in response to the salt treatment exhibited different trends according to the genotypes under study. Thus at the end of the treatment mature leaves of the salt sensitive Yuva contained higher amount of proline than those of Midyat. The changes in the calculated molar ratio between citrulline and free proline suggested that salt tolerance might be associated with high values for this ratio and vice et versa for sensitivity. The interest of citrulline as a biochemical marker for salt tolerance of melon genotypes is discussed.

scholarly journals The regulation of protein synthesis in the liver of rats. Mechanisms of dietary amino acid control in the immature animal

1968 ◽  
Vol 107 (5) ◽  
pp. 615-623 ◽  
Author(s):  
R. W. Wannemacher ◽  
W. K. Cooper ◽  
M. B. Yatvin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Specific Radioactivity ◽  
Protein Diet ◽  
Low Protein Diet ◽  
Deficient Diet ◽  
Low Protein ◽  
Rna Content ◽  
Cytoplasmic Rna

Weanling (23-day-old) rats were fed either on an amino acid-deficient diet (6% of casein, which in effect represents an ‘amino acid-deficient’ diet) or on a diet containing an adequate amount of protein (18% of casein) for 28 days. The hepatic cells from the animals fed on the low-protein diet were characterized by low amino acid content, almost complete inhibition of cell proliferation and a marked decrease in cell volume, protein content and concentration of cytoplasmic RNA compared with cells from control rats. The lower concentration of cytoplasmic RNA was correlated with a decreased ribosomal-RNA content, of which a larger proportion was in the form of free ribosomes. The protein-synthetic competence and messenger-RNA content of isolated ribosomes from liver cells of protein-deprived animals were 40–50% of those noted in controls. At 1hr. after an injection of radioactive uridine, the specific radioactivity of liver total RNA was greater in the group fed on the low-protein diet, but the amount of label that was associated with cytoplasmic RNA or ribosomes was significantly less than that noted in control animals. From these data it was concluded that dietary amino acids regulate hepatic protein synthesis (1) by affecting the ability of polyribosomes to synthesize protein and (2) by influencing the concentration of cytoplasmic ribosomes. It is also tentatively hypothesized that the former process may be directly related to the concentration of cellular free amino acids, whereas the latter could be correlated with the ability of newly synthesized ribosomal sub-units to leave the nucleus.

scholarly journals The Effect of Vincristine Sulphate on the Axoplasmic Flow of Proteins in Cultured Sympathetic Neurons

1973 ◽  
Vol 12 (2) ◽  
pp. 549-565
Author(s):  
J. M. ENGLAND ◽  
M. E. KADIN ◽  
M. N. GOLDSTEIN
Keyword(s):  
Amino Acid ◽  
Sympathetic Neurons ◽  
Metabolic Energy ◽  
Cell Protein ◽  
Amino Acid Incorporation ◽  
Axoplasmic Flow ◽  
Vincristine Sulphate ◽  
Morphological Differences ◽  
Cultured Sympathetic Neurons ◽  
The Relationship

The effect of vincristine sulphate on the axoplasmic flow of labelled proteins in neurites of chick embryo sympathetic neurons growing in tissue culture was studied by autoradiography. In control neurons most of the 3H-proteins synthesized during a 90-min pulse with a 3H-amino acid were localized in cell bodies. There was a diminishing gradient of labelled proteins in the neurites which was highest in portions adjacent to the cell bodies and lowest at the periphery. During a physiological chase there was a gradual increase in the amount of label in the neurites, so that after a 15-h chase even the most peripheral portions were well labelled. This indicates that a portion of the labelled proteins synthesized in the cell bodies are transported peripherally into the neurites. The centrifugal movement of labelled proteins in neurites was markedly decreased when cells were grown in medium containing 10 µg/ml vincristine sulphate. After a 15-h chase in the presence of drug only a small amount of label was in the peripheral portion of the neurites. Treatment with vincristine did not decrease the rate of amino acid incorporation or alter the rate of protein turnover during the course of the experiment. Thus an explanation of the results based on an altered rate of total cell protein synthesis or degradation is unlikely. The capacity of sympathetic neurons to take up and concentrate exogenous [3H]norepinephrine in their neurites was only slightly reduced by vincristine. This indicates that at least some cellular activities requiring metabolic energy are relatively unaffected by the interruption in axoplasmic flow caused by vincristine and that the mechanism by which vincristine interferes with axoplasmic flow does not involve general cellular toxicity. The major morphological differences between control and vincristine-treated neurons were the absence of microtubules and the presence of crystal-like structures within the cells. The relationship between the effect of vincristine on the axoplasmic flow of proteins and the arrangement of the microtubule system is discussed.

Whole body protein synthesis: studies with different amino acids in the rat

1989 ◽  
Vol 257 (5) ◽  
pp. E639-E646 ◽  
Author(s):  
C. Obled ◽  
F. Barre ◽  
D. J. Millward ◽  
M. Arnal
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Protein Turnover ◽  
Specific Radioactivity ◽  
Whole Body ◽  
Body Protein ◽  
Tissue Specific ◽  
Dose Method ◽  
Infusion Method

These studies were undertaken to determine to what extent constant infusion measurements and plasma sampling could provide sensible answers for rates of whole body protein turnover and also which amino acid would be the most representative probe of whole body protein turnover. Whole body protein synthesis rates were estimated in 70-g rats with L-[U-14C]threonine, L-[U-14C]lysine, L-[U-14C]tyrosine, L-[U-14C]phenylalanine, and L-[1-14C]leucine by either simultaneous tracer infusion of four amino acids or by injections of large quantities of 14C-labeled amino acids. In the infusion experiment, indirect estimates of whole body protein turnover based on free amino acid specific radioactivity and stochastic modeling were compared with direct measurement of the incorporation of the tracer into proteins. These two methods of analysis provided similar results for each amino acid, although in each case fractional synthesis rates were lower (by between 26 and 63%) when calculations were based on plasma rather than tissue specific radioactivity. With the flooding-dose method, whole body fractional protein synthesis rates were 41.4, 25.6, 31.1, and 31.4% with threonine, lysine, phenylalanine, and leucine, respectively. These values were similar to those obtained by the continuous infusion method using tissue specific radioactivity for threonine and lysine. For leucine, however, the flooding-dose method provided an intermediate value between the two estimates derived either from the plasma or the tissue specific radioactivity in the infusion method.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A Label-Free Assay for Aminoacylation of tRNA

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1173
Author(s):  
Howard Gamper ◽  
Ya-Ming Hou
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Cell Growth ◽  
Quantitative Measurement ◽  
Label Free ◽  
Radioactive Amino Acid ◽  
Protein Synthesis Machinery ◽  
Wide Range ◽  
Health And Disease

Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3′-32P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3′-32P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.

scholarly journals Protein Intake to Maximize Whole-Body Anabolism during Postexercise Recovery in Resistance-Trained Men with High Habitual Intakes is Severalfold Greater than the Current Recommended Dietary Allowance

2019 ◽  
Author(s):  
Michael Mazzulla ◽  
Sidney Abou Sawan ◽  
Eric Williamson ◽  
Sarkis J Hannaian ◽  
Kimberly A Volterman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Amino Acid ◽  
Resistance Exercise ◽  
Protein Intake ◽  
Whole Body ◽  
Total Amino Acid ◽  
Acid Oxidation ◽  
Amino Acid Oxidation ◽  
Habitual Protein Intake

ABSTRACT Background Dietary protein supports resistance exercise–induced anabolism primarily via the stimulation of protein synthesis rates. The indicator amino acid oxidation (IAAO) technique provides a noninvasive estimate of the protein intake that maximizes whole-body protein synthesis rates and net protein balance. Objective We utilized IAAO to determine the maximal anabolic response to postexercise protein ingestion in resistance-trained men. Methods Seven resistance-trained men (mean ± SD age 24 ± 3 y; weight 80 ± 9 kg; 11 ± 5% body fat; habitual protein intake 2.3 ± 0.6 g·kg−1·d−1) performed a bout of whole-body resistance exercise prior to ingesting hourly mixed meals, which provided a variable amount of protein (0.20–3.00 g·kg−1·d−1) as crystalline amino acids modeled after egg protein. Steady-state protein kinetics were modeled with oral l-[1-13C]-phenylalanine. Breath and urine samples were taken at isotopic steady state to determine phenylalanine flux (PheRa), phenylalanine excretion (F13CO2; reciprocal of protein synthesis), and net balance (protein synthesis − PheRa). Total amino acid oxidation was estimated from the ratio of urinary urea and creatinine. Results Mixed model biphasic linear regression revealed a plateau in F13CO2 (mean: 2.00; 95% CI: 1.62, 2.38 g protein·kg−1·d−1) (r2 = 0.64; P ˂ 0.01) and in net balance (mean: 2.01; 95% CI: 1.44, 2.57 g protein·kg−1·d−1) (r2 = 0.63; P ˂ 0.01). Ratios of urinary urea and creatinine concentrations increased linearly (r = 0.84; P ˂ 0.01) across the range of protein intakes. Conclusions A breakpoint protein intake of ∼2.0 g·kg−1·d−1, which maximized whole-body anabolism in resistance-trained men after exercise, is greater than previous IAAO-derived estimates for nonexercising men and is at the upper range of current general protein recommendations for athletes. The capacity to enhance whole-body net balance may be greater than previously suggested to maximize muscle protein synthesis in resistance-trained athletes accustomed to a high habitual protein intake. This trial was registered at clinicaltrials.gov as NCT03696264.

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