From the Department of Biochemistry, Haworth Hall, University of Kansas, Lawrence, Kansas 66045
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ABSTRACT |
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We have measured the in vivo protein turnover for the major calcium regulatory proteins of the sarcoplasmic reticulum from the skeletal muscle of young adult (7 months) and aged (28 months) Fischer 344 rats. From the time course of the incorporation and decay of protein-associated radioactivity after a pulse injection of [14C]leucine and correcting for leucine reutilization, in young rats, the apparent half-lives for calsequestrin, the 53-kDa glycoprotein, and ryanodine receptor are 5.4 ± 0.4, 6.3 ± 1.3, and 8.3 ± 1.3 days, respectively. A half-life of 14.5 ± 2.5 days was estimated for the Ca-ATPase isolated from young muscle. Differences in protein turnover associated with aging were determined using sequential injection of two different isotopic labels ([14C]leucine and [3H]leucine) to provide an estimate of protein synthesis and degradation within the same animal. The Ca-ATPase and ryanodine receptor isolated from aged muscle exhibits 27 ± 5% and 25 ± 3% slower protein turnover, respectively, relative to that from young muscle. In contrast, the 53-kDa glycoprotein exhibits a 25 ± 5% more rapid turnover in aged SR, while calsequestrin exhibits no age-dependent alteration in turnover. Statistical analysis comparing the sensitivity of various methods for discriminating different rates of protein turnover validates the approach used in this study and demonstrates that the use of two isotopic labels provides at least a 6-fold more sensitive means to detect age-related differences in protein turnover relative to other methods.
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INTRODUCTION |
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A general age-associated decline in the function of multiple proteins has been correlated with several types of posttranslational modifications, particularly oxidation, glycation, isomerization, deamidation, racemization, and cross-linking (1-3). Although the exact processes leading to the increase in these dysfunctional proteins remains undefined, it has been suggested that the greater accumulation of such modifications with age may result, at least in part, from the diminished capacity of the cell for the removal and replacement of defective proteins. In agreement with this hypothesis, total protein turnover in a number of tissues has been reported to decrease with age. However, a number of studies also report conflicting results, due perhaps to the complications inherent in measurements of total protein turnover, which represent weighted averages from a complex mixture of turnover rates (reviewed in Ref. 3).
In the present study, we have addressed the possibility that altered protein turnover may be responsible for the diminished skeletal muscle contractile properties observed with age that are directly attributable to SR1 proteins involved in excitation-contraction coupling, e.g. the Ca-ATPase and the calcium release channel (the ryanodine receptor) (4, 5). In the case of the Ca-ATPase, which mediates the rate-limiting resequestration of calcium into the SR lumen, oxidative modifications have been implicated in the observed age-related loss of heat stability and concomitant conformational alterations (6-8). Previous in vivo measurements of overall turnover for a mixture of SR proteins in microsomal preparations from young animals indicate a long half-life (10-14 days), suggesting the potential for considerable oxidative modification during the residence of one or more of these proteins in the cell (9). However, protein turnover rates for individual SR proteins during aging have not been determined.
Therefore, we have estimated the individual in vivo half-lives for several calcium regulatory proteins of the SR, i.e. the Ca-ATPase and the ryanodine receptor (RyR), as well as calsequestrin, a low affinity, high capacity calcium-binding protein, and the abundant SR protein, the 53-kDa glycoprotein, a putative regulator of the Ca-ATPase (10). Half-life estimates were made from the time-dependent incorporation and decay of protein-associated radioactivity after a single injection of [14C]leucine and monitoring free leucine in the serum to correct for leucine reutilization. As a more sensitive means to discriminate differences in protein turnover, relative rates of protein turnover were measured for young and aged Fischer 344 rats using the dual-isotope method, which uses two different isotopes of the same amino acid to provide an estimate of rates of protein synthesis and degradation within individual animals (11). The measured half-life for these calcium regulatory proteins, ranging from 5 to 14 days, confirm that these are long-lived proteins. Moreover we find that the most stable of these, the Ca-ATPase and the RyR, exhibit significantly slower turnover in aged skeletal muscle. We suggest that the decreased turnover of these key proteins controlling excitation-contraction of muscle may, in part, account for the increased accumulation of protein modifications and the diminished skeletal muscle performance observed in aged organisms.
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EXPERIMENTAL PROCEDURES |
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Materials
The following materials were purchased from the respective companies.
Antibodies-- Primary monoclonal antibodies to SERCA1, SERCA2a, calsequestrin, and triadin were from Affinity Bioreagents, Golden, CO; monoclonal antibody to RyR was a gift from the laboratory of Dr. John Sutko (University of Nevada); polyclonal antibody to phosphorylase b was from Biogenesis, Sandown, NH; secondary goat anti-mouse IgG-horseradish peroxidase-linked antibody was from Pierce.
Radioisotopes-- Uniformly labeled L-[14C]leucine and L-[3H](4,5)-leucine were from American Radiolabeled Chemicals, Inc., St. Louis, MO. Reagents for gel electrophoresis, electroblotting, and prestained molecular size markers were from Bio-Rad. MOPS, 4-chloro-1-naphthol, and all other reagents were from Sigma.
Animals
The animals used in this study were young adult (7 months,
396 ± 8 g) and aged (28 months, 426 ± 12 g)
Fischer 344 male rats obtained from the NIA colonies maintained at
Harlan Sprague-Dawley, Inc. Rats were housed separately during the
study and maintained under strictly controlled environmental conditions
for temperature (25 °C) and light/dark cycles (12 h intervals). Food
(NIH-31, Harlan Teklad Laboratory diet, Madison, WI) and tap water were provided ad libitum. Weight was monitored 2 days before the
first isotope injection for all rats, and again on day 5 for the rats involved in the dual-isotope procedure. The average net weight change
during this time was +1 ± 1 g and 5 ± 1 g for
young and aged rats, respectively. The research described in this
report was conducted in compliance with all applicable federal statutes and regulations relating to animals and adheres to the principles stated in The Guide for Care and Use of Laboratory Animals
(47).
Radioisotopes and Counting
Uniformly labeled L-[14C]leucine (specific activity 292 mCi/mmol) and L-[3H](4,5)-leucine (specific activity 60 Ci/mmol) were diluted with 0.9% normal sterile saline and administered by intraperitoneal injection at a (non-flooding) dose rate of 75 µCi/100 g body weight and 225 µCi/100 g body weight, respectively. To control for diurnal fluctuations in amino acid absorption, all isotope injections were administered at the same time (between 10:00 and 10:30 a.m.) each day.
Radioactivity from samples of serum, native SR preparations, and associated with protein bands on polyacrylamide gels was determined by scintillation counting using a Packard Tri-Carb liquid scintillation analyzer (model 1600 TR). Disintegrations per minute (dpm) was determined using a transformed external standard spectrum, which accounts for sample quenching and any resulting spectral distortion. For dual-isotope labeled samples, optimal radionucleotide separation was provided by the automatic region compensation method, which adjusts the spillover of each radionucleotide in the defined counting regions. Counting was carried to at least 2% accuracy.
Determination of Free Leucine in Serum
After sacrifice by CO2 inhalation, the chest cavity
was opened and blood was withdrawn from the heart. The blood was
centrifuged (3000 × g for 10 min) and the serum was
frozen at 70 °C for later amino acid analysis and counting of
radioactivity. Free leucine content in serum was determined as
described previously (12) after phenylisothiocyanate derivatization
using a Beckman (System Gold) amino acid analyzer (Biochemical Research
Services Laboratory, University of Kansas). Parallel samples were
assayed (in triplicate) for radioactivity by liquid scintillation
counting.
Isolation of SR Proteins
Native SR vesicles were isolated from rat hindlimb skeletal
muscles as described previously (13), with minor modifications (14). SR
vesicles were suspended in a medium consisting of 0.3 M
sucrose, 20 mM MOPS (pH 7.0), and stored at 70 °C.
Protein concentrations were determined by the method of Lowry using
bovine serum albumin as the standard (15). The amount of radioactivity in native SR vesicles (containing all SR proteins) was determined after
15 h of base hydrolysis (1 M NaOH) at 65 °C.
Hydrolyzed samples were neutralized with HCl before scintillation
counting (16).
Separation and Identification of SR Proteins
SR proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 5% separating gel with a 3% stacking gel, according to the method of Laemmli (17). To identify the location of specific proteins, Western immunoblotting was performed using monoclonal antibodies to SERCA1 (1:10,000), SERCA2a (1:250), calsequestrin (1:5000), triadin (1:5000), RyR1 (1:20), and the polyclonal antibody to phosphorylase b (1:500) (18). The secondary goat anti-mouse IgG-horseradish peroxidase-linked antibody was diluted 1:2000. Color development was accomplished using the peroxidase substrate 4-chloro-1-naphthol (3 mg/ml in methanol).
Antibodies to the 53-kDa glycoprotein are not commercially available. Therefore, this protein was identified by previous reports of apparent molecular mass based on its relative mobility on SDS-PAGE (19). In addition, identification of the 53-kDa glycoprotein was verified by the increased mobility of the 53-kDa protein band after cleavage of the carbohydrate moiety with endoglycosidase H, as outlined by Campbell and MacLennan (20).
The relative content of individual SR proteins in each native preparation was determined from densitometric measurements (Hewlett Packard ScanJet II scanner and Sigma Scan software) of 5% Laemmli SDS-polyacrylamide gels stained with Coomassie Blue. The amount of total SR protein (20 µg) used for determining relative percentages for individual proteins was within the linear range of detection for all proteins.
To determine the amount of radioactivity associated with each constituent protein, 1 mg of SR proteins were separated using a 5% SDS-polyacrylamide preparative gel (16 cm × 20 cm) (17). After electrophoresis, gels were stained with Coomassie Blue, and the protein bands previously identified by immunoblotting for specific SR proteins were processed and counted as described previously (16). Four to six separate measurements were made for proteins from each animal.
Leucine specific activity was calculated based on the leucine content reported in the published amino acid sequence of the RyR (985 nmol/mg of protein), the SERCA1a isoform of the Ca-ATPase (851 nmol/mg of protein), calsequestrin (937 nmol/mg of protein), and the 53-kDa glycoprotein (992 nmol/mg of protein) (21-24).
Half-life Determination from the Time Course of Protein-associated Radioactivity
As described previously for the estimate of half-lives of brain proteins, the time dependence of the incorporation and loss of radioactivity associated with serum leucine and SR proteins was measured after a single intraperitoneal injection of [14C]leucine into young adult (7 months) rats (n = 9) (25). Animals were sacrificed at sequential times ranging from 4 h to 13 days, SR membranes were isolated, and serum samples collected from each animal for scintillation counting as described above.
The time-dependent profile of serum leucine specific
activity was used to develop a series of theoretical curves for
proteins of different half-lives, according to the model of Zilversmit (26) as described previously (12). The serum data (from 4 h to 13 days) was best described by a three exponential curve, which represents
a 6-fold reduction in the reduced chi-squared (2R = 0.341) over a two-exponential model
(Fig. 2A). We note that residuals comparing the
three-exponential model to the experimental data were randomly
distributed. The function of specific radioactivity of the precursor
(F(t)) at early time points (0-4 h) was completed by
back-extrapolation of this three-exponential curve to a maximum serum
radioactivity of 720 ± 60 dpm at 3.0 ± 0.6 h and a
linear increase from 0 to 3 h provided the best fit to the protein
data (P(t)) (Fig. 2). Theoretical curves for proteins of
different half-lives were generated by numerical calculations according to Equation 1.
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(Eq. 1) |
dP/dt is the change in protein
radioactivity over time, P(t) is leucine specific
radioactivity associated with a protein at a given time, k
is the first order rate constant of degradation (which is equal to
0.693 divided by the protein half-life (t1/2)), and
F(t) is the specific radioactivity of the isotope at a given time in the precursor pool. These curves were fit to the data of the
time-dependent incorporation and decay of radioactivity associated with the RyR, the Ca-ATPase, calsequestrin, and the 53-kDa
glycoprotein (Fig. 2B). The best fit of the data to
theoretical curves was determined by 2 minimization
(27). The error in fits is provided at the 95% confidence limits.
Turnover Indices
For age-related comparisons of relative protein turnover, the dual-isotope labeling method was employed (11). Young adult (n = 11) and aged (n = 7) rats each received an intraperitoneal injection of 14C-labeled leucine, followed by a second injection of 3H-labeled leucine 4 h before sacrifice at 7, 11, or 15 days after 14C injection. The 4-h interval for exposure to the second isotope was chosen based on the profile of protein-associated incorporation and decay from 14C-injected young rats as a period of synthesis that is short relative to the time of peak isotope incorporation and thus minimal leucine reutilization is expected, but provides adequate radioactive signal. One young adult rat (control animal) received a simultaneous dose of both isotopes before sacrifice 4 h later to correct for the different amounts of radioactivity injected for the two isotopes.
The dual-isotope technique allows for the determination of relative turnover rates based on an estimate of both synthesis (from 3H) and degradation (from 14C), within the same animal. The 3H/14C ratios obtained from different times of sacrifice after 14C injection were not significantly different for any of the SR proteins examined, justifying the combination of ratio data for each protein from 7, 11, and 15 days. Differences between individual animals, e.g. lean body weight or size of their free amino acid pools, that would alter the effective isotope dosage were corrected for by dividing the isotope ratio obtained from each SR protein by the isotope ratio determined from the total of all SR proteins for each rat (28). This normalized ratio is referred to as the turnover index. In a similar manner, relative protein synthesis for individual SR proteins was determined by dividing the 3H radioactivity associated with individual proteins by the 3H radioactivity from total SR proteins for each rat (29).
Protein Half-life Determination
Estimates of protein half-lives were made for SR proteins from young and aged animals using three different methods.
Method 1-- The 3H/14C ratio was used to calculate a protein half-life as in Glass and Doyle (30). This method assumes that 3H represents the maximum level of radiolabel incorporated before decay begins. However, in these experiments, only a small fraction of the maximum amount of isotope was incorporated in 4 h into SR proteins. Using 3H dpm as an estimate of each individual's synthesis rate, the maximum radioactivity was predicted from the protein curves estimating half-lives for specific proteins from each age group (see Method 3 below). The 14C dpm were corrected for the unequal amount of radioactivity injected for the two isotopes by using the mean 3H/14C ratio for individual SR proteins (2.41 ± 0.08) isolated from the control animal, as a correction factor.
Method 2-- A decay constant (kd) was determined from linear regression analysis of the semi-log plot of the decay of 14C (ln dpm/nmol leucine) from 7 to 15 days for each protein. The slope of this line is a rough estimate of the first order degradation rate constant (kd) used to calculate the half-life (t1/2) using the expression: t1/2 = 0.693/kd (31). The standard error of the slope was used to estimate the error in half-life.
Method 3--
[14C] data from 7 to 15 days for
each protein was fit to protein curves which were generated from the
precursor data of serum leucine specific activity. The excellent fit of
14C-labeled free leucine from the data of young
single-isotope injected animals to the data from dual-isotope injected
young and aged rats (Fig. 2A) justifies utilizing
information from the single isotope experiment for these half-life
curve predictions. The goodness of fit of experimental data to a
theoretical half-life curve was determined by minimizing chi-squared
(2). The uncertainty in obtained fits to half-life
curves was calculated from the fit of experimental data to a parabolic
function of
2 near its minimum, as outlined by Bevington
and Robinson (27).
Calculation of the Limits of Sensitivity for Methods of Data Analysis
The minimum significant difference (d) that can be detected between groups is considered the limit of sensitivity of a method, and was calculated according to Equation 2.
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(Eq. 2) |
n is the number of individuals needed, s
is the standard deviation of the measurement, and t values
are the critical values from the Student's t distribution
for the probability values () indicated (P is the power
of the statistical test) for the appropriate degrees of freedom (32).
Probability values, i.e. defining the error in determining
significant differences, were set at 0.05 and the statistical power,
i.e. the probability of being able to find a difference of
this magnitude, was set at 0.75 (75% statistical power).
Alternatively, Equation 2 was rearranged, solving for n and
setting d to 20% of the mean experimental value, to
calculate the number of animals required to detect a 20%
difference.
Statistical Analysis
Differences between age groups were tested for statistical
significance using Student's t test analysis, with the
level of significance set at p 0.05. If the
assumption of the t test for equal variance between groups
was violated, results from the Aspin-Welch unequal variance
t test were used. The Fisher least significant difference
multiple comparison test was used to identify significantly different
turnover indices for individual SR proteins (p
0.05). Linear regression analysis was performed to determine the slope
and error associated with the fit of data for semi-log plots of the
natural logarithm of protein specific radioactivity as a function of
time. Slopes were considered significantly different if there was no
overlap in their 95% confidence intervals.
Data are reported as age-based group means ± S.E. A t-squared test for outliers, defined as observations that appear to be inconsistent with the majority of the data, was performed and where indicated, outliers were eliminated before data analysis. Analysis were performed on a personal computer using the Number Crunching Statistical System 6.0.1 for Windows (NCSS, Kaysville, UT) and Origin 4.1-32 bit (Microcal, Northampton, MA) software.
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RESULTS |
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Identification of SR Proteins by Western Immunoblotting-- SDS-PAGE was used to separate the constituent proteins in native SR membranes for the determination of radioactivity associated with the major calcium regulatory proteins in the SR. Western immunoblotting was performed to define the precise position on polyacrylamide gels of protein bands corresponding to the fast and slow twitch isoforms of the Ca-ATPase (SERCA1 and SERCA2a, respectively), triadin, phosphorylase b, the RyR, and calsequestrin (Fig. 1).
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Half-life Estimates of SR Calcium Regulatory Proteins-- The time dependence of incorporation and loss of radioactivity associated with serum leucine and with SR proteins was measured to determine the apparent half-lives of these SR proteins. Fig. 2 illustrates the relationship between the time-dependent disappearance of radioactivity in the blood (panel A) and the concomitant incorporation and decay of radioactivity in SR proteins (panel B). The measurable amounts of radioactivity present in the blood at 13 days after the injection indicates the need to account for leucine reutilization.
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Determination of Relative Protein Turnover in Young and Aged Rats-- The dual isotope method has been successfully used to determine differences in protein turnover between experimental groups (28, 36). Individual (7 and 28 month old) animals were exposed to both [14C]leucine for 7, 11, or 15 days, and to [3H]leucine, administered 4 h before sacrifice for all animals. Relative rates of protein synthesis and degradation were estimated from the isotope ratio of 3H/14C measured for each SR protein from individual animals (11). The resulting isotope ratio was used to calculate a turnover index by normalizing the 3H/14C ratio for each protein with the isotope ratio obtained from all SR proteins for that individual animal (28). This normalization factor does not change with age of the animal. The mean isotope ratio for all SR proteins calculated for the group of 7-month-old animals is 2.2 ± 0.1 and for the group of 28-month-old animals, 2.3 ± 0.3. Normalization in this manner accounts for variation in lean muscle weight relative to total body weight (Table II) and the resulting differences in the effective isotope dose, thereby reducing the variability between individuals and consequently increasing the ability to discriminate differences between groups.
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Estimates of Synthesis Rates-- The extent of [3H]leucine (dpm) incorporation at 4 h after injection for each SR protein, normalized with the 3H accumulated in all SR proteins for each animal, provides an estimate of relative protein synthesis rate (Table III). The relative synthesis rate for individual SR proteins was identical (within the 95% confidence limits), irrespective of whether the first isotope (14C) was administered 7, 11, or 15 days before the second isotope (3H) (data not shown). Therefore, data from different sample times were combined for each age group. Comparing the relative rates of protein synthesis, we find that the Ca-ATPase isolated from aged skeletal muscle has a significantly slower synthesis rate than Ca-ATPase protein from young muscle (p = 0.003). No significant age-related difference in the relative rate of synthesis is observed for any of the other SR proteins examined, implying that differences in protein turnover of the RyR and the 53-kDa glycoprotein result mainly from altered rates of degradation.
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Power Analysis of Analytical Methods for Determining Protein Turnover-- One of the most important aspects of designing experiments is consideration of the sensitivity of the method of measurement and the number of individuals included. These considerations are essential to the ability to detect group differences. Therefore, we have used these experimental data to conduct a power analysis of several methods that have been frequently used to determine protein turnover to provide guidelines for those who may wish to pursue similar investigations.
Power analysis of our data was used to evaluate the effectiveness of the turnover index for the detection of age-based differences in protein turnover, and to compare this method with other methods commonly used to calculate protein half-lives, including (i) the use of the ratio data as described previously, (ii) fitting semilog plots of the time-dependent decay of protein-associated 14C to a linear regression, and (iii) using serum leucine data to generate theoretical curves for proteins with different half-lives (as in Fig. 2) (Table IV). In agreement with previous suggestions, we observe that the turnover index provides a substantially greater sensitivity for discriminating differences between groups compared with other methods of analysis (11). For example, turnover indices can detect a 4-16% difference between experimental groups for the Ca-ATPase, while other methods are 2-14-fold less sensitive. The method that provides the least power to determine differences is the linear regression fit of semi-log plots of the time-dependent decay of protein-associated 14C. Moreover, these results confirm that the number of animals used in this study was sufficient to adequately investigate relative rates of turnover. Also apparent from this analysis is that the greater variability associated with the aged animals dictates that larger numbers of these animals must be utilized to discriminate group differences.
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DISCUSSION |
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Summary of Results-- We have measured the in vivo turnover rates of individual calcium regulatory proteins from skeletal muscle SR and examined their protein turnover as a function of age. We find that the proteins, the Ca-ATPase, the RyR, calsequestrin, and the 53-kDa glycoprotein, are all long-lived; apparent half-lives for the RyR, calsequestrin and the 53-kDa glycoprotein range from 5 to 8 days, and 14 days for the Ca-ATPase, as determined from the decay of [14C]leucine after a pulse injection and correcting for leucine reutilization. Using the dual-isotope method, which minimizes individual variability, we observe that the rates of relative protein turnover are altered in a protein-specific manner in aged rats. An approximate 25% decrease in protein turnover is observed for both the RyR and Ca-ATPase, and a 25% increase in turnover of the 53-kDa glycoprotein from aged rats. No age-related alteration in relative turnover rate was observed for calsequestrin. A power analysis of the different methods used to determine protein turnover confirms the validity of our approach with respect to both the use of turnover indices as a sensitive means to detect age-related differences and the number of animals used in this study.
Discussion of Assumptions and Analytical Methods-- For the dual-isotope method, a number of assumptions are implicit (11). These assumptions include that (i) at the time of sacrifice, the isotope administered first has begun to decay, and (ii) the isotope administered second has not yet reached peak incorporation. A third assumption is that after accumulation of the maximum amount of isotope, the loss of protein-associated radioactivity follows exponential decay kinetics. These assumptions were verified by monitoring multiple time points throughout the incorporation and decay of radiolabeled SR proteins after a single injection of [14C]leucine in young rats. The decay of protein-associated radioactivity was exponential and from this profile, appropriate times were selected for which all SR proteins were in the synthesis phase for 3H accumulation and in the decay phase after 14C administration. A fourth assumption is that the rates of protein synthesis are the same at the time when the first and second injections are administered. We have addressed this consideration by administering injections at the same time of day for both isotopes to minimize diurnal fluctuations in protein synthesis. This assumption is validated by the similar amount of tritium incorporated into individual SR proteins within each age group, irrespective of whether the 3H injection was administered along with [14C]leucine (control animal), or at 7, 11, or 15 days after the 14C administration. Finally, it is assumed that the isotope is not metabolized to different products that can be incorporated into the measured protein fraction. This assumption has been addressed with the use of leucine, which has been previously demonstrated not to be significantly metabolized to other amino acids in liver and skeletal muscle (11, 38).
Physiological Relevance-- Knowledge of the cellular residence time for individual proteins provides insight into the feasibility of potential posttranslational modifications, as well as allowing for assessment of potential biochemical mechanisms responsible for these alterations. Liver proteins, for which the majority of half-life data is available, have been classified into groups of proteins with (i) very fast turnover (half-life less than 1 h), (ii) fast turnover (half-life 1-24 h), or (iii) slow turnover (half-life over 24 h) (reviewed in Ref. 39). Even accounting for the 2 to 5 times slower turnover of skeletal muscle, the SR calcium regulatory proteins examined in this study can be considered proteins with slow turnover. Two other ion transporters, the plasma membrane Ca-ATPase from the brain and the Na+-K+ ATPase from kidney, have similarly lengthy half-lives, i.e. 12 days and 4 days, respectively (25, 40). Thus, proteins with slow turnover have considerable potential for accumulation of most posttranslational modifications during their residence time in the cell. Perhaps the only modification that has been implicated in aging that is unlikely to occur to any significant extent is the Amadori rearrangement (non-enzymatic glycosylation) involved in protein glycation, which requires several weeks under conditions found in the cell (41).
In view of the significantly longer lifetime observed for both the Ca-ATPase and the RyR from aged skeletal muscle, we postulate that these two proteins are prone to a greater accumulation of modifications with age. The observed identical age-dependent decrease in the relative turnover rates of these two SR proteins that function in tandem, i.e. sequester and release calcium in the SR, is consistent with their coordinate regulation reported under a variety of conditions in both cardiac and skeletal muscle. These include the coordinate induction of biosynthesis of both proteins during muscle differentiation, and their coordinate regulation in response to pharmacological or physiological manipulation (42-45). In contrast, turnover of calsequestrin was not altered as a function of age, suggesting that regulation of cellular concentrations of this protein is not under the same control as either the Ca-ATPase or the RyR. Previous studies have reported that calsequestrin turnover, in both skeletal or cardiac muscle, does not respond in a similar manner as the Ca-ATPase and RyR as a result of aging in the heart, cardiac hypertrophy, or exposure to cold temperatures (44-46).Significance of This Study-- In this study, we observe that the effect of aging on protein turnover in skeletal muscle SR was specific to individual proteins; the RyR and Ca-ATPase experience slower relative rates of turnover, whereas the 53-kDa glycoprotein turned over more quickly in aged rats, and no change in calsequestrin turnover between age groups was measured. In contrast, from comparison of the 3H/14C ratios for all SR proteins, we find no age-related changes in overall turnover. These results emphasize the importance of examining individual proteins as opposed to mixtures of proteins when investigating aging effects. The information gained from measuring the turnover of a mixed protein population is limited because it provides only an average value for the turnover rate. The significant changes in turnover of specific proteins that may occur with age could be masked by other proteins that are found in greater abundance or have significantly different half-lives.
Based on the results presented in the current investigation, decreased protein turnover of the Ca-ATPase and RyR in aged skeletal muscle could account, in part, for the diminished skeletal muscle function and alterations in the Ca-ATPase protein reported previously (4, 5, 7). However, we cannot rule out that aging may also be accompanied by changes in the cellular environment that result in greater exposure of proteins to oxidizing conditions and, in turn, a general increase in oxidized proteins. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. James Bresnahan, Terry Jones, and Nancy Schwarting for help with isotope injections, and Michael Lemon from the Office for Radiation Safety for advice with the handling of radioactive substances. Drs. Jack Schlager and Peter Reeds provided invaluable advice.
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FOOTNOTES |
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* This work was supported by grants from the American Federation for Aging Research (AFAR) for a Glenn/AFAR Scholarship (to D. A. F.) and Grants RO1 AG12275 and PO1 AG12993A from the NIA, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 785-864-3831;
Fax: 785-864-5321.
1 The abbreviations used are: SR, sarcoplasmic reticulum; dpm, disintegrations per minute; PTS2, phosphorylase b, triadin, and SERCA2a; RyR, ryanodine receptor; SERCA1, fast twitch Ca-ATPase isoform; SERCA2a, slow twitch Ca-ATPase isoform; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic acid.
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REFERENCES |
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