Leucine Pools in Normal and Dystrophic Chicken Skeletal Muscle Cells in Culture*

The specific radioactivity of [3H]Leu in the extracel- lular, intracellular, and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307) embryonic chick breast muscle cultures was analyzed as a function of equilibration time and extracellular Leu concentra- tion (0.05-5 mM). The primary results were the following 1) r3H]Leu equilibrated to a constant specific radio- activity in the intracellular and Leu-tRNA pools within 2 min after addition to both normal and dystrophic cultures. 2) After equilibration, the extracellular [3H] Leu specific radioactivity in dystrophic cell culture medium was lower than that of medium exposed to normal cells (especially at low Leu concentrations), probably because of increased release of unlabeled Leu from the dystrophic cells as a result of faster protein breakdown. Accordingly, the specific radioactivities in the intracellular and the Leu-tRNA pools were also lower in dystrophic cells. 3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA pool was approximately 40% lower than the specific radioactivity in the intracellular pool in both normal and dystrophic cells. Thus, high concentrations of extracellu- lar Leu cannot be used to “flood out” reutilization of unlabeled Leu (released by protein degradation) during protein synthesis. 4) At 5.0 mM extracellular Leu, the specific radioactivity of in the intracellular was comparable to in the extracellular

The specific radioactivity of [3H]Leu in the extracellular, intracellular, and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307) embryonic chick breast muscle cultures was analyzed as a function of equilibration time and extracellular Leu concentration (0.05-5 mM). The primary results were the following 1) r3H]Leu equilibrated to a constant specific radioactivity in the intracellular and Leu-tRNA pools within 2 min after addition to both normal and dystrophic cultures. 2) After equilibration, the extracellular [3H] Leu specific radioactivity in dystrophic cell culture medium was lower than that of medium exposed to normal cells (especially at low Leu concentrations), probably because of increased release of unlabeled Leu from the dystrophic cells as a result of faster protein breakdown. Accordingly, the specific radioactivities in the intracellular and the Leu-tRNA pools were also lower in dystrophic cells. 3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA pool was approximately 40% lower than the specific radioactivity in the intracellular pool in both normal and dystrophic cells. Thus, high concentrations of extracellular Leu cannot be used to "flood out" reutilization of unlabeled Leu (released by protein degradation) during protein synthesis. 4) At 5.0 mM extracellular Leu, the specific radioactivity of [3H]Leu in the intracellular pool was comparable to that in the extracellular pool in normal and dystrophic cells; however, the specific radioactivity of Leu-tRNA (Le. the immediate precursor to protein synthesis) was only 55-65% of the extracellular specific radioactivity in normal and dystrophic cells. In conclusion, reutilization of Leu from protein degradation is higher in dystrophic muscle cell cultures than in normal muscle cell cultures, and accurate rates of protein synthesis in cell cultures can only be obtained if specific radioactivity of amino acid in tRNA is measured.
One of the early defects in muscular dystrophy may be an alteration in plasma membrane structure and function. Dystrophic muscle shows increased permeability to enzymes and ions (Herman and Fernandez, 1977;Weinstock and Jones, 1977) and exhibits a number of morphological alterations associated with the plasma membrane (Mokri and Engel, *This work was supported by Grants AM30823 and AM01095 from the National Institute of Health and Grant PRM8120163 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Supported by a postdoctoral fellowship from the Muscular Dystrophy Association.
§ To whom correspondence should be sent.
1975 ;Schotland et al., 1977). Other membrane functions, such as amino acid transport, may also be affected in dystrophic cells, and these changes could lead to alterations in cellular amino acid pools and ultimately muscle protein metabolism. Most studies of protein metabolism employ radiolabeled amino acids. Unfortunately, many invalid assumptions about the specific radioactivity of the precursor pools and about the reutilization of amino acids have been made. Recently, several investigators have measured protein synthesis by analyzing the specific radioactivity of amino acids bound to tRNA, and hence the immediate precursor to protein synthesis (Airhart et al., 1979;Hildebran et al., 1981;Martin et al., 1977;McKee et al., 1978;Schneible and Young, 1981). These studies convincingly illustrate the need for accurate measurements to ensure the validity of using amino acid data to calculate true rates of synthesis. Airhart et al. (1979) andMcKee et al. (1978) have proposed models for compartmentation of amino acids for protein synthesis within the transport system of the cell membrane and therefore link membrane function intimately to the protein metabolism mechanisms within the cell. This possibility is especially intriguing in relationship to genetic muscular dystrophy where changes in both the cell membrane and protein breakdown may occur. Thus, the primary goal of this study was to determine the relationships among the extracellular, intracellular, and Leu-tRNA pools in skeletal muscle cells cultured from normal and dystrophic muscle. These relationships were studied both to examine possible alterations caused by muscular dystrophy and to enable more accurate measurements of the rates of protein synthesis and breakdown in normal and dystrophic muscle cells. A preliminary report of some of these results has appeared previously (Schneible and Young, 1981).

EXPERIMENTAL PROCEDURES
Experimental Design-The relationships among cellular amino acid pools were examined by studying the specific radioactivity of Leu in the pools at various times after administration of [3H]Leu and by examining the effect of Leu concentrations on the steady state Leu specific radioactivity. Cell culture of muscle from normal and dystrophic chick embryos was chosen as the experimental system because the culture medium surrounding the cells could be manipulated easily to examine the relationships among amino acid pools and because 7-8-day-old muscle cell cultures are at steady state with respect to myofibrillar protein synthesis and degradation. Extracellular, intracellular, and Leu-tRNA pools were investigated. The extracellular pool was defined as the quantity of free Leu in the cell culture medium, the intracellular pool was defined as the free Leu that was within the cytoplasm but not attached to tRNA, and the Leu-tRNA pool was defined as the quantity of Leu attached to tRNA and presumably the direct precursor to protein synthesis.
Muscle cells were cultured in 85% Eagle's minimum essential medium containing 10% horse serum, 5% chick embryo extract, 50 units/ml of penicillin, 50 pg/ml of streptomycin, 2.5 pg/ml of Fungizone, and 85 pg/ml of gentamycin. Prior to initiation of each experi-

Leucine Pools in Dystrophic Muscle
Cultures 1437 ment, cells were incubated in unlabeled experimental medium (85% Eagle's minimum essential medium without Leu, 10% horse serum, 5% embryo extract, M fluorodeoxyuridine, and various concentrations of Leu) for 30 min to allow cellular amino acid pools to equilibrate with that particular medium. [4,5-'H]Leu was then added in a small volume of buffered saline, and the kinetics of equilibration of this newly added [3H]Leu into cellular pools was followed.
Cell Culture-Fertilized eggs from line 307 genetically dystrophic chickens (Wilson et al., 1979) were obtained from the Department of Avian Sciences, University of California, Davis. Fertilized white leghorn eggs used as the controls were obtained from Feather Crest Hatchery, Arkadelphia, AL. Primary cultures of skeletal muscle were prepared by the procedure of Young et al. (1981). Embryonic breast muscle was dissected from 13-day-old embryos. The tissue was disaggregated into individual cells by vortexing the suspension on a Vortex mixer for 30 s, and cells were recovered by centrifugation. The cells were counted in a hemocytometer and plated at approximately 4 X lo6 cells/6O-mm collagen-coated tissue culture plate. Culture medium was changed every 24 h, and the cells were incubated at 37 "C in a 5% COz atmosphere. When muscle cell proliferation began to decline (usually day 3 of culture), M fluorodeoxyuridine was added to the culture medium to inhibit fibroblast proliferation. CUItures were examined after 6-8 days, at which time the cells were at steady state with respect to protein synthesis and degradation (Young et al., 1981).
Specific Radioactivity of Cellular Leu Pools-After incubation with ['HILeu for various times, the medium was removed from the plate and saved for measurement of extracellular specific radioactivity as described later. Cells were washed by successive immersion in five beakers, each containing 200 ml of ice-cold buffered saline.
Intracellular free Leu and Leu bound to tRNA were isolated and analyzed for specific radioactivity as detailed by Airhart et al. (1979). Briefly, the cells were lysed in 0.5 ml of a buffered detergent solution, and a 0.2-ml aliquot was removed to determine intracellular amino acid specific radioactivity. The remaining solution was extracted with phenol, and nucleic acids were precipitated by addition of ethanol. Amino acids were dissociated from tRNA by base hydrolysis, and the amino acid solution was evaporated to dryness under vacuum (Airhart et al., 1979).
The portion of the cell lysate set aside for intracellular amino acids was treated immediately with 5 volumes of 10% (w/v) trichloroacetic acid. The solution was cooled to 4 "C and the precipitated protein was pelleted by centrifugation. This pellet was used for measurement of ['Hlleucine incorporation into protein (see below). The supernatant, containing free amino acids, was extracted eight times with ether to remove trichloroacetic acid. A sample was evaporated to dryness for determination of Leu specific radioactivity in the intracellular pool. Similarly, the cell culture medium was deproteinized by addition of 100% (w/v) trichloroacetic acid to a final concentration of lo%, the precipitated protein was pelleted by centrifugation, and trichloroacetic acid was removed from the supernatant by five extractions with ether. The extracellular samples were not evaporated, however, because the amino acid concentration was high enough for direct measurement of specific radioactivity.
The dried samples of aminoacyl-tRNA and intracellular amino acids were taken up in bicarbonate buffer as described by Airhart et al. (1979) to a final pH of 8.5-10. A small aliquot of the deproteinized labeling medium was also brought to pH 8.5-10 with this buffer. All samples were then reacted with ["C]dansyl' chloride, and two-dimensional chromatography was performed on Cheng Chin micropolyamide thin layer plates (Airhart et al., 1979). The Leu spot was cut out, and the 3H and 14C disintegrations/min were determined by liquid scintillation spectrometry. Since the [3H]Leu was reacted with ["C] dansyl chloride, the ratio of the two isotopes in the resulting dansylleucine could be used to measure the specific radioactivity of the Leu by the following formula: SA Leu = (3H dpm/14C dpm) (SAoNs,,) (K), where SA = specific radioactivity in disintegrations/min/pmol, DNS C1 = dansyl chloride, and K = moles of dansyl chloride bound per mol of amino acid and equals 1 for Leu.
Radiolabeled Leu Incorporation into Protein-The precipitated protein from the intracellular fraction was filtered through glass fiber filters. The filters were washed with 19 ml of 5% trichloroacetic acid and 5 ml of 95% ethanol, dried, and placed in scintillation vials. NCS tissue solubilizer (0.5 ml) was added, and the vials were heated a t ' The abbreviation used is: dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. 50 "C for at least 1 h to solubilize protein. After the vials were cooled to room temperature, radioactivity was measured by liquid scintillation spectrometry.
Cell Counts-Replicate muscle cell cultures in 60-mm tissue culture plates were stained with Giemsa stain as described by Young et al. (1975). At least 600 nuclei were counted in a minimum of five randomly chosen fields. The number of nuclei within multinucleated myotubes, the number of mononucleated cells, and the percentage of nuclei within multinucleated myotubes were calculated and used as indices of growth and differentiation of muscle cell cultures and as a normalization factor for incorporation of ['HILeu into protein.
Measurement of radioactivity was performed in a Beckman liquid scintillation spectrometer. The Leu spots from the micropolyamide plates were placed into 7-ml glass minivials (Wheaton Scientific), and 0.15 ml of NCS and 3.0 ml of 3a20 scintillation fluid were added.
Samples were allowed to equilibrate at least 12 h before analysis to minimize the background counts/min. Glass fiber filters were counted in 10 ml of 3a20 scintillation mixture. Efficiencies of counting and isotope spill in dual isotope experiments were monitored using the external standard channels ratio method with chloroform as the quenching agent. Average efficiencies were 40% for 14C, 32% for 3H, and 18% spill of "C into the 3H channel for samples containing both isotopes. Efficiency was approximately 40% for 3H analyzed alone.
Materials-Embryo extract was prepared from homogenates of decapitated 12-day-old white leghorn chick embryos. Gentamycin and collagen were from Sigma, and fluorodeoxyuridine was obtained from Calbiochem-Behring. Other tissue culture media and reagents were from Grand Island Biological Co. (Grand Island, NY). Tissue solubilizer (NCS) and [3H]Leu were from Amersham Corp., and 3a20 scintillation mixture and [14C]dansyl chloride were purchased from Research Products International Corp. (Mt. Prospect, IL). Cheng Chin micropolyamide thin layer plates and nonradioactive dansyl chloride were from Pierce Chemical Co. Glass fiber filters (Whatman GF/C) were obtained from Reeve Angel (Clifton, NJ). Phenol (J. T. Baker Chemical Co., Phillipsburg, NJ) was redistilled prior to use in prot.ein extractions. intracellular, and Leu-tRNA pools of normal and dystrophic muscle cultures in medium with a Leu concentration of 0.05 mM, the lowest concentration used in this investigation. In both cases, the [3H]Leu was added at zero time to the culture medium; thus, the extracellular [3H]Leu specific radioactivity attained its highest value immediately upon initiation of the experiment. Because the quantity of Leu in the extracellular pool was large compared to the quantity in the other two pools, its specific radioactivity diminished only 10-15% during the 30-min incubation period. This decrease in extracellular specific radioactivity presumably was caused by the release of nonradioactive amino acids from cellular protein by protein degradation. In both normal and dystrophic cultures, the intracellular and Leu-tRNA specific radioactivities reached a steady state within 2 min and remained essentially constant thereafter. Thus, ["]Leu was rapidly distributed to various pools within the muscle cell, and the dystrophic condition had no apparent effect on the time course of this distribution. Additional measurements at 60 and 120 min after addition of ["]Leu showed that the specific radioactivity in all three pools was not significantly different from the values observed in Fig. 1 after 30 min (data not shown).

Uptake of Radiolabeled Leu into Cellular
In both normal and dystrophic cultures, the equilibrated specific radioactivities of the intracellular and Leu-tRNA pools were lower than the extracellular specific radioactivity (Fig. 1, A and B ) . The intracellular and Leu-tRNA pools receive Leu from both the external medium and degradation of endogenous protein. Because the Leu given off by protein degradation is not radioactively labeled and because some of this nonradioactive Leu is reutilized for synthesis of new proteins, the specific radioactivities of these two pools are lower than that of the extracellular pool. This condition should persist until an equilibrium is reached between the rate of [3H]Leu incorporation into protein (i.e. when all proteins are uniformly labeled) and the rate of release of ["HI Leu by protein degradation. Although the time required for uniform protein labeling was not measured, results of experiments carried out for up to 2 h were identical with the distributions shown in Fig. 1 after 30 min.
If A and B of Fig. 1 are compared, one primary difference between normal and dystrophic cultures is observed. The values for extracellular Leu specific radioactivity in dystrophic cultures were lower by approximately 45% compared to those in normal cultures, even though the same quantity of [3H]Leu was added to the medium bathing both cell types. For example, the specific radioactivity of [3H]Leu in the extracellular pool of normal cells after 30 min was 998 dpm/ pmol (Fig. l A ) , but only 531 dpm/pmol in the extracellular pool of dystrophic cells after 30 min (Fig. 1B). The values for steady state intracellular and Leu-tRNA specific radioactivity in dystrophic cells were also proportionally decreased compared to normal cultures. The best explanation for the lower extracellular specific radioactivity in dystrophic cells even a t zero time after addition of [3H]Leu is a consequence of the experimental design (see "Experimental Procedures"). Prior to addition of ["]Leu directly to the cultures, the cells were preincubated for 30 min in culture medium containing the appropriate concentration of nonradioactive Leu. This action was taken to allow the cells to adjust to the altered extracellular Leu concentration before the ["]Leu was added. In dystrophic cells, where protein degradation was apparently faster, a larger quantity of nonradioactive Leu was released during the 30-min preincubation period, and this faster release was then manifested as a lower extracellular specific radioactivity immediately upon addition of I3H]Leu.

Steady State Specific Radioactivities In Cellular Leu Pook-
The effect of extracellular Leu concentration on the equilibrated specific radioactivity of cellular [3H]Leu pools is shown in Table I. Note that the values for specific radioactivity decrease in all cases as the Leu concentration was increased. This occurred because the quantity of [3H]Leu was not increased as a constant proportion of the total quantity of Leu in the cell culture medium. Three practical considerations dictated that a cmstant extracellular specific radioactivity could not be employed over the range of 0.05-5.0 mM Leu. First, the cost of conducting adequate numbers of experiments with 5 mCi/ml of 13H]Leu would have been prohibitive. Second, 5 mCi/ml of ["]Leu is a sufficiently high level of radioactivity to risk radiation toxicity to the cells and thereby induce aberrant results. Third, the embryo extract and horse serum components of cell culture medium both contain small quantities of nonradioactive Leu. This exogenous Leu would have prevented rigorous definition of [3H]Leu specific radioactivity, especially at 0.05 mM leucine. For these reasons, direct comparisons of data in Table I can be made only among samples analyzed at the same medium Leu concentration. Comparison among the different Leu concentrations is described later.
As expected from data in Fig. 1, the extracellular specific radioactivity in dystrophic cultures was considerably lower than that in normal cultures at 0.05 mM Leu (Table I). At 0.2 mM Leu, the dystrophic extracellular [3H]Leu specific radioactivity was only slightly lower than the normal value; but at 5.0 mM Leu, the difference was unexplainably large again. Additionally, the intracellular and Leu-tRNA specific radioactivites in dystrophic cultures are lower than the corresponding values in normal cells a t all three Leu concentrations (Table I). Table I1 also shows the effect of Leu concentration on cellular Leu pools; however, the specific radioactivities of the equilibrated intracellular and Leu-tRNA pools from Table I have been recalculated as percentages of the corresponding extracellular [3H]Leu specific radioactivies. Recalculation in this manner more clearly illustrates a crucial point. As the extracellular Leu concentration was increased, the intracellular [3H]Leu specific radioactivity values approached the values for the extracellular Leu pool. Stated differently, extracellular Leu contributed more and more Leu to the intracellular pool as the external Leu concentration was raised, and the proportion of intracellular free Leu coming from protein breakdown was concomitantly decreased. At the highest Leu concentration studied (5.0 mM), the intracellular specific radioactivity equaled the extracellular specific radioactivity in normal cells and was only slightly lower in dystrophic cells.
In contrast to the intracellular pool, the specific radioactivity of the Leu-tRNA pool was only minimally responsive to changes in Leu concentration (Table 111, implying that the proportions of amino acids from the extracellular pool and from protein degradation were not significantly affected. Therefore, high extracellular Leu does not appear to eliminate or even significantly alter reutilization of amino acids from breakdown of cellular proteins as is frequently assumed. Indeed, the fact that Leu-tRNA specific radioactivity in both normal and dystrophic cells was only 55-60% of extracellular specific radioactivity implies that 40-45% of the Leu incorporated into muscle protein originates from reutilization.

Total Protein Synthesis Rate in Dystrophic
Muscle Cultures-To critically compare protein synthesis in normal and dystrophic cultures, muscle cells were pulse-labeled for 2 h with 0.1 mCi/ml of [3H]Leu in complete culture medium containing a physiological concentration of Leu (0.2 mM).

TABLE I Effect of leucine concentration on [3Hlleucine specific radioactivity
Cultured cells from normal and dystrophic muscle were exposed to medium containing [ 3H]Leu at the indicated concentrations. The specific radioactivities of cellular Leu pools were measured 30, 60, or 120 min after addition of [3H]Leu (0.05 mM, 50 pCi/ml; 0.2 mM, 100 pCi/ml; 5.0 mM, 500 pci/ml). As indicated in the text, specific radioactivities measured at either 30, 60, or 120 min were not significantly different, and the data from all three times were therefore pooled for this table. Data are expressed as mean & S.E. from 2 to 12 experiments with multiple samples.
The concentration of Leu listed reflects only that added as nonradioactive Leu. The actual Leu concentration was higher because of the Leu in horse serum and embryo extract. Assuming the concentration of Leu in serum and embryo extract is approximately 0.3 mM and that these components make up 15% of the final volume of the culture medium, the actual concentrations of Leu in the labeling media would have been approximately 0.095, 0.245, and 5.045 mM. The calculated [3H]Leu specific radioactivities at each of these three extracellular Leu concentrations prior to exposure to cell cultures would therefore have been approximately 1160,900, and 220 dpm/ pmol, respectively.
Data from dystrophic cells were compared to their normal counterparts in each cellular compartment using a t test. * indicates p < 0.05; ** indicates p < 0.01.
e Means for normal cells in the same row not bearing the same superscript are different from each other at the 0.05 level using a t test. Note that this comparison is only made within each row of data for each concentration of Leu.
Means for dystrophic cells in the same row not bearing the same superscript are different from each other at the 0.05 level using a t test.
e" Refers to means that are compared according to the conditions in Footnotes c and d. TABLE I1 Leucine specific radioactivity Cultured cells from normal and dystrophic muscle were exposed to medium containing [3H]leucine at the indicated concentration. The specific radioactivity of cellular leucine pools was measured (see Table  I). Data are expressed as the mean f S.E. from two to seven experiments with multiple samples in each experiment. Data are per cent of extracellular specific radioactivity.

Intracellular
Leu conc.   111, line 3). Third, conversion of the incorporation data (or apparent synthesis rates) into actual protein synthesis rates using Leu-tRNA specific radioactivity revealed that total protein synthesis was approximately 42% higher in dystrophic cells than in normal cells. Because muscle cell cultures are at a steady state with respect to protein synthesis and degradation after 7-8 days and because the synthesis rate must equal the degradation rate at steady state, the data in Table I11 also indicate that protein degradation is approximately 42% higher in cultured dystrophic cells.

DISCUSSION
The amino acids that are polymerized into peptide chains can originate from either the extracellular environment as a result of active amino acid transport or from reutilization of amino acids that were liberated by proteolysis of pre-existing proteins. When the relative contribution of amino acids from these two sources is unknown, it is difficult to accurately measure rates of protein synthesis or breakdown. Our previous research (Riebow and Young, 1980;Young et al., 198l), which suggested that myofibrillar protein synthesis and breakdown were elevated in dystrophic muscle cells, employed pulse label and pulse-chase techniques. These data were therefore subject to the following assumptions. 1) The contribution to protein synthesis of amino acids from reutilization was negligible compared to the contribution from active transport, and 2) normal and dystrophic muscle cells derive the same fraction of amino acids from reutilization and transport. From Tables I and 11, it is clear that neither of these assumptions was completely valid. For example, in normal and dystrophic cells, the tRNA specific radioactivity was 55% of the extracellular specific radioactivity, implying that approximately 45% of the amino acids for protein synthesis originated from reutilization. Moreover, the specific radioactivity of tRNA in dys-trophic cells is less than the specific radioactivity of tRNA in normal cells (Table I).
In contrast to the differences reported here for line 307 dystrophic chick cultures, Wolitsky et al. (1982) have found no differences in protein synthesis and degradation in cultured muscle cells from line 412 normal and 413 dystrophic muscle cultures. Line 412 is a better genetic control for line 413 than the white leghorn chick is for line 307 dystrophic chickens, and some of the differences reported here undoubtedly reflect genetic divergence unrelated to the dystrophic lesion. In any case, discovery of the mechanism by which protein degradation is accelerated so drastically in line 307 cells should provide interesting insights into the mechanisms by which protein metabolism can be altered in muscle disease or muscle atrophy.
One of the most important points illustrated by this study concerns accurate calculations of protein synthesis and degradation rates in cultured muscle and in muscle explants. The assumptions are frequently made that elevation of extracellular amino acid concentration "floods out" reutilization of nonradioactive amino acids from protein degradation and that the specific radioactivity of the intracellular pool can be assumed to be equal to the specific radioactivity of the tRNA pool. Similar assumptions are made when pulse-chase experiments are used to investigate protein degradation. The present study shows that intracellular specific radioactivity does equal extracellular specific radioactivity a t high concentrations of Leu; however, the Leu-tRNA specific radioactivity is unresponsive to extracellular Leu concentration and is a maximum of 55-60% of the extracellular specific radioactivity. A directly comparable analysis has been reported when phenylalanine specific radioactivity was investigated in pulmonary macrophages (Hammer and Rannels, 1981), and these observations have been confirmed in other cell types (Hildebran et al., 1981;.. Negligence in measuring the tRNA specific radioactivity could therefore lead to errors of up to 35-40% in quantitating protein synthesis or degradation rates in cell cultures. The possibility that calculated rates of protein synthesis using Leu-tRNA specific radioactivity contain additional errors other than those discussed above cannot be eliminated. An inherent assumption is that all isoaccepting species of Leu-tRNA are randomly charged from the extracellular pool and from protein degradation and that these isoaccepting species are then randomly utilized for protein synthesis. If different isoaccepting species of Leu-tRNA were to receive their amino acid from unique pools within the cell, measurement of the averaged specific radioactivity of these isoaccepting species would inaccurately reflect the specific radioactivity of the individual species. Direct proof that this does not occur can only be obtained by comparing the specific radioactivity in tRNA with the specific radioactivity within nascent chains of specific proteins. In at least one reported instance, indirect evidence indicates that selective amino acid pool utilization does not take place (Hildebran et al., 1981). The model of interaction of proline pools in human lung cells illustrated by Hildebran et al. (1981, Fig. 4) may be generally applicable to other amino acids and is useful in visualizing the complexity of intracellular amino acid pools.
Muscle atrophy is characteristic of muscular dystrophy, and changes in protein metabolism must occur to explain this loss of muscle tissue. Many studies have described such changes but differ in whether the change in metabolism is due to a change in synthesis, degradation, or both (Battelle and Florini, 1973;Riebow and Young, 1980;Weinstock et al., 1969;Young et al., 1978 and. The underlying key assumption of studying muscular dystrophy in cell culture is that the genetic lesion is expressed independently of physical or biochemical contact with other dystrophic tissues or organs (i.e. via nerve or blood). Several major lines of evidence support this assumption for genetic muscular dystrophy of the line 307 dystrophic chick (discussed by Young et al., 1981). Even though muscle cell cultures artificially represent the in uiuo situation, evidence for abnormalities in the proteolytic systems that metabolize myofibrillar proteins is reasonably convincing (Askanas et al. 1971;Battelle and Florini, 1973;McConnell et al., 1981, a and b;Goldberg et al., 1977;Riebow and Young, 1980;Rourke, 1975;Weinstock and Jones, 1977;Young et al., 1978 and.