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3 Donald W. Reynolds Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR 72205 and 4 Metabolism Unit, Shriners Burns Hospital and Department of Surgery, University of Texas Medical Branch, Galveston, TX 77550
* To whom correspondence should be addressed. E-mail: rwolfe2{at}uams.edu.
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ABSTRACT |
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Introduction |
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Limited studies suggest that tRNA are generally highly charged with amino acids under normal physiological conditions. However, the tRNA for only a few specific amino acids have been investigated. Charging of leucyl-tRNA has been found to be close to complete in the livers of rats, even after 1 or 2 d of starvation (11,12). However, there are no data available regarding the extent of charging of the tRNA for most amino acids in liver. Only indirect information regarding the extent of charging of tRNA in muscle is available. It has been reported that the Km (Michaelis constant, defined as that concentration of substrate that gives "half-maximal enzyme activity") for the synthase enzyme responsible for the charging of leucyl-tRNA in rat muscle is well below the normal intracellular concentration (13). To our knowledge, there is no information regarding the relation between the Km and corresponding concentrations of intracellular amino acids other than leucine. Further, there are no data quantifying the actual charging of tRNA for any amino acid in muscle.
We have developed a new approach to fully understand the possible role of precursor availability in controlling the rate of protein synthesis in vivo. Rather than focus on the percentage charging of tRNA, we have instead measured the total availability of charged tRNA, because this should be the more relevant parameter when considering precursor availability. Also, rather than focus on a single amino acid, we measured the availability of 16 aminoacyl-tRNA, because the synthesis of a protein requires adequate availability of all of the component amino acids. We used this methodology to determine differences in the total pool sizes of charged aminoacyl-tRNA in liver and muscle. The fractional synthetic rate of liver protein is several-fold greater than that of muscle (14). If there is a relation between precursor availability and protein synthesis, it should be reflected by a significantly greater aminoacyl-tRNA pool in liver than in muscle.
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Methods |
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Male New Zealand White rabbits (n = 6) (Myrtle's Rabbitry), weighing between 4 and 5 kg, were used for this study. The rabbits were housed in individual cages and acclimated with Lab Rabbit Chow HF 5326 (Purina Mills) and water for 1 wk. After overnight food deprivation, an amino acid mixture (10% Travasol; Baxter Healthcare) was i.v. infused at 0.5 mL–1·kg–1·h–1 under general anesthesia. One hundred milliliters of the Travasol solution contained the following amounts of the L-isomers of amino acids: 730 mg leucine, 600 mg isoleucine, 580 mg lysine hydrochloride, 580 mg valine, 560 mg phenylalanine, 480 mg histidine, 420 mg threonine, 400 mg methionine, 180 mg tryptophan, 2.07 g alanine, 1.15 g arginine, 1.03 g glycine, 680 mg proline, 500 mg serine, and 40 mg tyrosine. The infusion was given to maintain constant amino acid concentrations throughout the study. Three hours after the start of the infusion, we took fresh tissues from the liver and adductor muscle of hind limbs. Tissue samples were rinsed quickly in ice-cold saline (0.9% sodium chloride solution), frozen immediately in liquid nitrogen, and were stored in –80°C for further processing.
The anesthetic and surgical procedures have been described in previous publications (15,16). In brief, the rabbits were anesthetized with ketamine and xylazine. Polyethylene catheters (PE-90; Becton-Dickinson) were inserted in the right femoral artery and vein through an incision in the groin. The arterial line was used for blood collection and monitoring heart rate and mean arterial blood pressure; the venous line was used for infusion of amino acids. A tracheal tube was placed via tracheotomy. We placed the free end of the tracheal tube in an open hood that was connected to an oxygen supply line so that the rabbits spontaneously breathed room air enriched with oxygen.
This protocol complied with NIH guidelines and was approved by the Animal Care and Use Committee of The University of Texas Medical Branch at Galveston.
Aminoacyl-tRNA isolation
Aminoacyl-tRNA was isolated from 5 g of tissue using the A mirVanaTM miRNA Isolation kit (Ambion) (17). Ten 0.5-g pieces of tissue were processed separately and the tRNA pooled. To eliminate free amino acids that were not bound with tRNA, the tRNA pellet was washed with Wash Solution (provided by the mirVana) 3 times and resolved in nuclease-free water. The amount and purity of tRNA were assessed by measuring the absorbance at UV-260 nm (A260) and the ratio of A260:A280 in a DU650 spectrophotometer (Beckman Coulter). After determining the A260 value by multiplying the spectrophotometer reading by the dilution factor, the RNA concentration was calculated as described in the mirVana miRNA isolation instruction manual. As a check for purity, we analyzed the isolated tRNA fraction by electrophoresis on denaturing 15% polyacrylamide gel with 8 mol/L urea.
Aminoacyl-tRNA deacylation
Aminoacyl-tRNA was deacylated in 0.12 mol/L KOH, pH 9.0, at 37°C for 1 h, as reported by Davis et al. (18). Amino acids were separated from tRNA by acidification with 0.5 mol/L HCl to pH 2.0 and centrifuged at 3000 x g; 30 min. The supernatant, which contained amino acids, was dried under nitrogen gas stream. The dried amino acids were reconstituted in 1.0 mL of 1 mol/L acetic acid and 20 µL of a standard solution containing norvaline (10 µmol/L) and ß-aminobutyric acid (30 µmol/L). The amino acids were purified through a cation exchange column (Ag 50W-X8 resin, 200–400 mesh, H+ form, BioRad Laboratories) and dried in a speed vacuum concentrator (AES 2010–220, Savant Instruments) at room temperature. To measure the amino acids isolated from the aminoacyl-tRNA sample, 20 µL of acetonitrile and 80 µL of water was added to the dried amino acids. After setting on ice for 30 min, 60 µL of the sample was passed through an ultrafree-MC centrifugal filter (5,000 NMWL filter units, Millipore) by centrifuging at 4000 x g; 4 h at 4°C. The filtrates were stored at –80°C before HPLC analysis.
Isolation of blood free amino acids and protein-bound amino acids
To determine plasma free amino acid concentrations, 50 µL of arterial plasma was precipitated with 100 µL of cold acetonitrile. An aliquot of 100 µL of standard solution containing norvaline (10 µmol/L) and ß-aminobutyric acid (30 µmol/L) was added. After centrifugation, the supernatant was transferred to centrifugal filtration vials (5000 NMWL filter unit; Millipore) and centrifuged at 3000 x g; 5 h. The clear solution that had passed through the filter was used for HPLC analysis of amino acid concentrations. Tissues (liver or muscle) were hydrolyzed by 3 mL of 6 mol/L HCl at 110°C for 24 h. After hydrolysis, the tissue-bound amino acids of tissues were processed in the same manner as amino acids deacylated from aminoacyl-tRNA.
HPLC analysis of amino acid concentrations
The amino acid concentrations from plasma, the deacylated tRNA and hydrolyzed muscle and liver proteins were measured by reverse phase HPLC equipped with fluorescence detector using o-phthaldehyde derivative (18). The concentration of each individual amino acid was obtained from the chromatogram peak area comparison with standard. The amount of each individual amino acid bound with tRNA was calculated by measured concentration (nanomoles per milliliter) x 0.12 mL (the recovery factor), where 0.12 mL is the volume of each sample.
Method validation
tRNA recovery test. To evaluate tRNA recovery, the following experiment was repeated 4 times: 0.4 g of fresh rabbit liver was homogenized in 4 mL of lysis/binding buffer. The homogenate was evenly divided into 2 aliquots. 150 µg of tRNA (Type XI, from bovine liver, 50 units, 17.2 A260 units/mg, Sigma-Aldrich, cat. no. R-4752) in 100 µL water was added to aliquot B and 100 µL of nuclease-free water was added into aliquot A. They were then mixed and incubated 10 min on ice and the same aminoacyl-tRNA isolation procedure as described above was followed for both aliquots. The tRNA recovery was evaluated by the difference between the amounts of tRNA in each aliquot, divided by the amount of tRNA added.
Determination of possible contamination of tRNA pellet with free amino acids. The following 2 tests were performed to determine whether the measured amino acids were all derived from the aminoacyl-tRNA pool rather than the free amino acids in the tissue fluid.
In the first test, 0.9 µL of L-[4, 5-3H]-leucine (5.66 TBq/nmol, 153 Ci/mmol, cat no. TRK510, Amersham Biosciences) was mixed with the homogenization buffer before it was added to the frozen muscle tissue. The tissue was processed by the same RNA isolation procedure as described above. At each step of isolation and washing, 100 µL of solution was taken and measured by a LKB 1219 liquid scintillation counter (Wallac Oy).
In the 2nd test, 265 µmol of exogenous phenylalanine was added to 5 mL of the homogenization buffer before adding the frozen muscle tissue. All the discarded solutions were collected for measurement of phenylalanine in the solutions by HPLC. The concentration of free phenylalanine in the final discarded washing solution was compared with that of the corresponding value from the deacylation of charged tRNA.
Reproducibility and reliability of aminoacyl- tRNA isolation. To determine the reproducibility of the method, 10 pieces of muscle samples (5 g each) from 5 rabbits were processed for tRNA isolation and purification on 2 different days. The amount and purity of tRNA was assessed by the absorbance at UV-260 nm (A260) and the ratio of A260/280.
Statistical analysis
All values were expressed as means ± SEM. Statistical analysis between paired samples from muscle and liver were performed with 2-tailed paired t test. The same statistical approach was used to compare the fractional contribution of individual amino acids to the aminoacyl-tRNA pool and the corresponding muscle protein-bound pool. Significance was accepted at the level of P < 0.05.
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Results |
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Recovery of tRNA. Recovery was calculated as the difference between the samples before (A) and after (B) the standard was added; the means for aliquot A and B were 100.06 ± 0.46 and 182.14 ± 1.35 µg. Because the amount of standard added was 150.43 ± 0.74 µg, the mean recovery was 100 x (B – A)/150.43 = 54.6 ± 0.60%. Calculation of tRNA concentrations were therefore corrected for recovery.
Contamination of tRNA-bound amino acids with free amino acids. The radioactivity of the 3H-leucine added was 386.6 ± 3.95 Bq. After washing 3 times with the wash solution from the kit, the radiolabel in the discarded washing solution and tRNA pellet both declined to the background level. Therefore, there were no detectable free amino acids in the tRNA pellet. However, the absence of detectable radioactivity does not exclude the possible presence of a small undetectable amount of free amino acids. Therefore, a 2nd test was performed to compare the amounts of amino acids in the aminoacyl-tRNA pool and in the discarded washing solution. Results showed that the concentration of phenylalanine in the 3rd washing solution was 0.08 µmol/L, which was a minor percentage of the starting concentration (53 µmol/L) of free phenylalanine in the homogenization buffer. The contamination of the isolated tRNA with free amino acids was therefore considered to be acceptable.
Reproducibility and reliability of purification of tRNA. Repetitive analysis of muscle samples from 5 rabbits showed that there was consistent tRNA yield, with a CV of 7.5%. The A260:A280 ratio was 1.99 ± 0.01, indicating a high purity of the tRNA isolated as well. Visualization of the results of electrophoresis indicated only minor contamination with rRNA (Fig. 1).
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20% of the aminoacyl-tRNA pools in both tissues, but a minimal amount of either amino acid was present in the protein bound pool of muscle or liver, although albumin contains 11% of aspartate + asparagine (Table 2). The glutamine + glutamate aminoacyl-tRNA pool in muscle was significantly smaller than the protein-bound pool, but the values did not differ in liver. Conversely, the alanine aminoacyl-tRNA pool constituted >30% of the muscle pool compared with <10% of the muscle-bound pool of alanine (Table 2). In the EAA, the proportions of isoleucyl- and leucyl-tRNA were lower than their corresponding contribution to muscle protein whereas the proportions of histidyl- and lysyl-tRNA were greater than their contribution to muscle protein (Fig. 3). In the liver, the greatest discrepancies between the charged tRNA and protein-bound amino acid occurred for leucine and threonine. In the case of leucine, the proportional contribution of the aminoacyl-tRNA pool of EAA was only 12.8 ± 2.3% compared with 24.1 ± 4.2% in the protein-bound EAA pool (P < 0.05). Threonine, on the other hand, comprised 35.6 ± 3.2% of the aminoacyl-tRNA pool of EAA, but only 16.6% of the protein-bound EAA pool (P < 0.05). Histidine was significantly more abundant in the liver aminolacyl-tRNA pool than in the protein-bound pool and the converse was true for lysine. The distribution of EAA in the constitutive proteins of liver was similar to that of albumin (Fig. 3).
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Discussion |
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4 times the tRNA in the liver per gram of tissue than in muscle and that under the conditions of this experiment, the aminoacyl-tRNA pool was 7 times greater in liver. The charging percentage of aminoacyl tRNA was also determined in total tRNA. We further found that whereas the proportionate availability of certain species of aminoacyl tRNA was well matched with the corresponding contribution of that amino acid to the protein in that tissue, in other cases there were wide discrepancies.
To our knowledge, this article reports tissue levels of tRNA, as well as the individual aminoacyl-tRNA pools, for the first time. The 3 primary types of RNA molecules are mRNA, rRNA, and tRNA. tRNA comprises 12% of total cellular RNA (19,20). In most articles investigating the aminoacyl-tRNA pool, the total RNA has been isolated, rather than the tRNA specifically (e.g. 21–23). In this article, we have reported the isolation of tRNA from the mRNA and most of the rRNA. This isolation method enabled for the first time the quantification of the amount of charged tRNA in tissues. Because we have used a new method, it is pertinent to examine its validity.
The most likely source of error in the measurement of the aminoacyl-tRNA pool is contamination of the charged tRNA with free amino acids from the intracellular pool. There are 2 potential sources of contamination: exogenous contamination from the reaction system and endogenous amino acid contamination from free amino acids. A blank control was run to exclude exogenous interference. The radioactivity assay confirmed that there was no free amino acid in the purified aminoacyl-tRNA fraction. In addition, the 2nd contamination test involving the addition of exogenous phenylalanine showed that after the washing steps, the retention of exogenous amino acid was minimal. Further, the replicate analysis of 10 samples of muscle showed the procedure for measuring the amount of tRNA to be consistent and reproducible and the gel electrophoresis results documented good isolation of tRNA from mRNA and fairly complete isolation from rRNA.
The charging of tRNA generally is not considered to be rate limiting for protein synthesis (e.g. 23). However, the data directly supporting that conclusion are limited. Studies have shown that charging of leucyl-tRNA in liver is close to complete, even in the fasting state (11,12,18), and that deprivation of a single amino acid from the diet (isoleucine) does not affect the charging of the corresponding aminoacyl tRNA in the brain (13). However, a simultaneous assessment of the charging of several aminoacyl tRNA in these tissues has not been undertaken. Data are even more limited in muscle. The Km for the synthetase to form leucyl-tRNA is well below the intracellular concentration (13) and therefore it has been considered that the charging of tRNA is always complete in muscle as well (23). The extent of charging of tRNA in muscle with leucine, or any other amino acid, has not been measured.
The data presented in this article do not directly address the issue of the percent charging of the individual tRNA. However, if we assume that the molecular weight of tRNA is 2.5 x 104 (24), and the measured liver tRNA charging was 14.21 ± 4.42 nmol amino acids/mg tRNA and muscle charging was 7.00 ± 1.5 nmol amino acids/mg tRNA, it can be calculated that charging was 35.6% in liver and 17.5% in muscle. These data are inconsistent with previous reports of close-to-complete charging of leucyl-tRNA in liver (11,12,18) and muscle (13,23). The low extent of charging of total tRNA could be due to low charging of aminoacyl tRNA other than leucyl-tRNA. For example, a portion of the discrepancy between liver and muscle may be explained by the amount of glutamate + glutamine bound to tRNA in liver compared with muscle. In any case, the low values for total charging that we have observed give reason to examine in the future the extent of charging of individual tRNA.
There is indirect evidence that the extent of charging of tRNA does not control the rate of muscle protein synthesis. Thus, we have shown that muscle protein synthesis was stimulated during the infusion of amino acids into normal human subjects at rates sufficient to increase plasma concentrations within the normal physiological range, even though the intracellular concentrations of free amino acids remained either unchanged or slightly depressed (2). Because the amino acids involved in charging of muscle tRNA apparently come from the intracellular pool (7,8), our results are consistent with the notion that a stimulation of muscle protein synthesis above the normal basal rate is not mediated by an increase in tRNA charging. On the other hand, the potential role of the total availability of charged tRNA in regulating the rate of protein synthesis must be considered. Anderson (25) first suggested in 1969 that the total amount of tRNA may play an important role in regulating the rate of protein synthesis based on results from Escherichia coli. However, to our knowledge, this concept has not been extended to the in vivo situation. In this study, we found that the liver contained 3-fold more tRNA per gram of tissue than muscle and the amount of amino acids per gram of liver tRNA was also greater than in muscle, meaning that the amount of amino acids in the aminoacyl-tRNA pool in liver was 8-fold greater than in muscle. Garlick et al. (14) reported than in rat liver, the mean fractional synthetic rate of protein was 50% per day, which was 7-fold that of muscle (7.2% per day). It thus appears that amount of tRNA in a tissue, combined with the extent of charging, may be directly related to the rate of protein synthesis in that tissue. This observation suggests that not only is amino acid availability important but also that factors regulating the amount of tissue tRNA are important in determining the rate of synthesis of protein in a given tissue.
The profile of amino acids in the aminoacyl-tRNA and protein-bound pools reveal some interesting discrepancies that may relate to regulatory roles of individual amino acids. A number of studies, including our own, have pointed to a potential regulatory role of leucine in both whole body as well as muscle protein synthesis (26,27). In both liver and muscle, the amount of leucyl-tRNA was found to be disproportionately lower than the relative contribution of leucine to protein produced in that tissue (Fig. 3). Therefore, it is possible that the regulatory role of leucine stems from the relatively low amount of leucyl-tRNA in the basal state, thereby making leucine availability rate limiting. This speculation is consistent with our work in identifying the optimal formulation of amino acids to stimulate muscle protein synthesis, as we have found that a mixture containing a disproportionate amount of the branched chain amino acids to be advantageous compared with the distribution of amino acids in a high quality protein such as whey (28). Consequently, it may be that a formulation directed toward balancing the relationships between the aminoacyl-tRNA pools and the distribution of amino acids in protein produced in that tissue may be a means to specifically target the production of certain proteins.
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FOOTNOTES |
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2 Author disclosures: R. R. Wolfe, J. Song, J. Sun, and X. Zhang, no conflicts of interest.
5 Abbreviations used: EAA, essential amino acid; mRNA, messenger RNA; tRNA, transfer RNA.
Manuscript received 15 January 2007. Initial review completed 6 March 2007. Revision accepted 16 August 2007.
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