Stagewise decline in the activity of brain protein synthesis factors and relationship between this decline and longevity in two rodent species

Stagewise decline in the activity of brain protein synthesis factors and relationship between this decline and longevity in two rodent species

Mechanisms of Ageing and Development, 36 (1986) 197-210 197 Elsevier Scientific Publishers Ireland Ltd. STAGEWlSE DECLINE IN THE ACTIVITY OF BRAIN ...

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Mechanisms of Ageing and Development, 36 (1986) 197-210

197

Elsevier Scientific Publishers Ireland Ltd.

STAGEWlSE DECLINE IN THE ACTIVITY OF BRAIN PROTEIN SYNTHESIS FACTORS AND RELATIONSHIP BETWEEN THIS DECLINE AND LONGEVITY IN TWO RODENT SPECIES

MARIO CASTA~IEDA*, ROCIO VARGAS and SILVIA C. GALV,h,N Department of Developmental Biology, Instituto de Investigaciones Biom~dicas, UniversidadNacional Aut6noma de M~xico, 04510 M~xico, D.F. {Mdxico)

(Received May 13th, 1986) SUMMARY The activities of brain initiation factor 2 and brain elongation factor 1, which function as rate-limiting in total protein synthesis, and estimations of brain weight were followed during postnatal life in the rat and the mouse. Both activities decreased in parallel while cumulative brain weight increased. Three exponential components were required for the mathematical expression of each of the three processes in semilogarithmic plots against time. The acceleration curves for the activities and tissue weight demonstrated a mirror image symmetry. Within the general pattern of diminution with age, the negative acceleration of the activities and the positive acceleration of the brain weight displayed repeated bursts. The activities of both factors could also be arranged into several regression lines in log/log plots against time. Significantly, in these plots, the regression line calculated for the whole set of data for each factor activity showed that the value of the ratio of the slopes (mouse to rat) was inversely related to the square root of the ratio of species longevity and was in agreement with the power law relating life spans of cells to species longevity (R6hme, Proc. Natl. Acad. ScL U.S.A., 78(1981) 5009).

Key words: Rodent brain;Protein-synthesisfactors; Stagewise decay;Longevffy-related

decay INTRODUCTION That the capacity of mammalian brain to synthesize protein diminishes with age has been documented by using in vivo and cell-free systems from young and senescent organisms [1,2]. Significantly, this same change has recently been reported in human fibroblasts aged in vitro, and it has also been suggested that: (a) the reduced ability of these fibro*To whom correspondence should be addressed. 0047-6374/86/$03.50 Printed and Published in Ireland

© 1986 ElsevierScientificPublishers Ireland Ltd.

198 blasts to respond to growth factors is more a consequence of an unresponsive protein biosynthesis pathway than a defect in the action of growth factors [3] ; and (b) a defect in protein synthesis may compromise the availability of glycolytic enzymes which appear to be necessary for lymphocyte transformation [4]. Therefore, since this decreased capacity appears to be a basic phenomenon of aging, the study of the mechanism(s) of protein synthesis regulation is relevant. From the data obtained in different laboratories on cell-free systems, the candidates for being the most important components with this putative regulatory role have been narrowed to ribosomes and enzymatic translation factors. Reports trying to discriminate between these two components have been contradictory. Although the same or different species or organ systems have been used in these works, such discrepancies appear to be due mainly to the use of ribosome preparations contaminated with translation factors. Recently, two groups have reported the presence of defective ribosomes in aged organisms. In one, in which senile mouse liver was used, 40 S subunits were shown to have only a 10-20% decrease in activity in the initiation step assay; initiation factors were not tested [5]. In the other report, monosomes from an artificially aged nematode showed reduced activity in the phenylalanine polymerization assay [6]. Since 1971, several workers have shown that in the case of rodent brain (mainly rat and mouse;see ref. 2 for review), the activity of the ribosomes from brains of aged animals was comparable to that from newborn and young animals. In this organ system, Castafieda and his group found similar data for ribosomes and have shown that brain elongation factor 1 (bEF-1) and brain initiation factor 2 (bIF-2) both become qualitatively different during aging, function as rate-limiting activities, and are responsible (probably in conjunction with other initiation factors involved in the blF-2 assay) for the age-related decrease in activity observed in the cell-free systems [2,7]. Furthermore, upon inactivation, the blF-2 preparations from older brains behaved as a multicomponent system; since IF-2 has biosynthetic activity and is known to be phosphorylable, regulation through protein kinases was proposed [8]. The activity of EF-1 has also been reported to diminish in the above-mentioned nematode and rat liver during aging [9,10], serum-deprived cells [11], muscle of protein-deprived animals [12], and liver cirrhosis [13]. This same activity is also capable of increasing and appears to be rate-limiting in sea urchin eggs upon fertilization [ 14,15], spleen during the immune response [16], tissue-specific amino acid polymerization rates [17], liver during temperature acclimation [18], livers of tumor-bearing rats [19], and induction of hepatic detoxifying enzymes [20]. In our first report on rodent brain [2], from the data obtained on bEF-1 from the very first months of postnatal life we thought that a simple exponential model would explain the decay of activity as a function of age. However, for the present work, we had access to an older population of animals and extended the period of analysis. We found that the rate of decay of bEF-1 and blF-2 activities: (a) changes intermitently with time; and (b) is inversely related to brain mass, to species longevity, and to the life spans of cells in vitro and in vivo.

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POSTNATAL AGE (doys) Fig. 1. Age-dependent variation in the activities of blF-2 and bEF-1 (right-hand ordinates) and in wet brain weight (left-hand ordinates) of the rat (panel A) and mouse (panel B), Point values represent the mean of triplicate assays from three independent experiments. The S.E.M, values for all the points were <15% and w e r e not depicted for clarity. Each panel contains its own ordinate and abscissa scales. Reaction mixtures, processing, counting, and weight estimations were as described in Materials and Methods.

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201 MATERIALS AND METHODS Tissue and subcellular fractions Animals (Wistar rats and Swiss albino mice), tissues, ribosomal-wash protein (RWP), ammonium sulfate fractions, [aSS] Met-tRNA i, and elongator aminoacyl-tRNA (AA-tRNA) were obtained and prepared as previously described [2,7]. Briefly, after groups o f animals o f the same postnatal age were decapitated, their brains were removed, weighed, and then rinsed with and homogenized in ice-cold 50 mM Tris-HC1 (pH 7.4), containing 35 mM KC1, 5 mM magnesium acetate, 1 mM dithiothreitol (DTT) and 350 mM sucrose in a glassTeflon tissue grinder. Tissues from animals o f at least three different ages were processed in parallel for comparative purposes. Each homogenate was sequentially centrifuged, at 4°C, at 15 0 0 0 , 30 000, and 150 000 g for 15, 15, and 120 min, respectively. The last supernatant was adjusted to pH 6.5 with acetic acid and the upper two-thirds o f the resulting supernatant was subjected to ammonium sulfate fractionation and was dialyzed as described by McKeehan and Hardesty [21]. This dialyzed fraction was treated with 10 mM N-ethylmaleimide to differentially inhibit brain elongation factor 2 and was used as a source of bEF-1 [2]. The ribosomal pellet was rinsed and resuspended in 50 mM TrisHCI (pH 7.4), containing 35 mM KCI, 1 mM DTT, and 10% glycerol. The suspension was made 0.5 M with respect to KCI and then centrifuged at 150 000 g for 120 min at 4°C. The upper four-fifths of this supernatant was used to obtain the RWP which served as a source o f initiation factors [6]. Protein concentration was determined by the method o f Lowry et al. [22]. Initiator and AA-tRNA were prepared from total rat liver tRNA. One portion of this preparation was radiolabelled by charging the tRNA with t-lasS] methionine (1000 Ci/ mmol, New England Nuclear) by aminoacyl-tRNA synthetase from Escherichia coli under conditions for the initiator species [23]. The [aSS] Met-tRNAi obtained in the reaction mixtures was purified by ion-exchange chromatography on a DEAE-cellulose (Whatman DE-23) column with 1 M KC1 as the eluting solution. The procedure described by Moldave [24] was used to charge the other portion of total tRNA with a mixture o f amino acids, with a radioactively labelled amino acid used as a monitor.

Fig. 2. Semilogarithmic plot of mean activities of factors and tissue weight as a function of age for the rat (A) and mouse (B). Original data for this and the following figures were taken from Fig. 1. The abscissa scales for the activities of factors are the same but have been displaced to the right 60 and 80 units for bIF-2 and bEF-1, respectively, for clarity purposes. Also, the points for day 540 in panel A are off the abscissa scale. The regression lines (numbered from left to right), of the general form y = b + rex, were obtained by the method of least squares with the following b, m, r parameters and P values: rat brain weight Yl, 2.25, 0.054, 0.99, <0.001;y~, 2.99, 0.001, --0.98, 0.01 ;y~, 3.15, 0.0003, 0.81, 0.01; rat blF-2 y,, 5.97, --0.03, --0.99, <0.001; y~, 3.69, --0.015, -0.96, <0.001; Ya, 5.1, -0.00001, -0.34, >0.1; rat bEE-1 Yl, 3.69, -0.015, -0.96, <0.001; Y2, 3.52, -0.003, -0.99, <0.001; y~, 3.1, -0.00005, -0.95, 0.01; mouse brain weight Yl, 1.8, 0.06, 0.95, <0.001,y2, 2.4, 0.002, 0.98, <0.001; Ya, 2.56, -0.0001, -0.84, 0.05; mouse blF-2 Yl, 6.1, -0.04, -0.97, <0.001, y~, 5.5, -0.0036, -0.94, 0.01 ; Ya, 5.29, -0.003, -0.99, 0.01 ; mouse bEF-1 yl, 4.23, -0.047, -0.99, <0.001;y 2, 3.67, -0.002, -0.96, <0.001;y~, 3.53, -0.0003, --0.81, 0.1, respectively.

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POSTNATAL AGE (doys) Fig. 3. Age-dependent variation in the deceleration o f the activities of blF-2 and bEF-I of the rat (A) and m o u s e (B). T h e absolute values of the decrements between each date of m e a s u r e m e n t (1 or more days) were taken as positive n u m b e r s and were divided by the n u m b e r of days elapsed during the interval. T h e logarithm of the resulting value was used for the ordinate scales for clearer presentation. lnsets: Age-dependent variation in the increments of brain weight and in the decrements of bEF-1 activity (calculated on per day basis) of the rat (A) and m o u s e (B).

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Enzymatic binding assays The activities of both blF.2 and bEF.1 were measured under zero-order conditions for the substrates so that the systems were dependent only on the activity of the preparations of these factors. The formation of the Met-tRNA i. bIF-2 • GTP and AA-tRNA, bEF-I. GTP ternary complexes, respectively, were used to measure these activities as previously described and validated [2,7]. The reaction mixture (0.1 ml) for bIF-2 contained 50 mM Tris-HC1 (pH 7.4), lO0 mM KC1, 1.5 mM GTP, 1 mM DTT, 3 mM phosphoenolpyruvate, l0/ag pyruvate kinase, 20/,tg [3SS]Met-tRNAi ('q).4 /aCi), and around 100 pg RWP which has been shown to be the saturating concentration for this system (see Figs. 2A and 3 or ref. 7). After incubation at 37°C for 15 rain, the reaction was stopped with 3 ml of an ice-cold mixture of 20 mM Tris-HCl (pH 7.4) and 100 mM KC1. The resulting mixture was passed through a nitrocellulose filter (0.45 /am pore size, Millipore) which, in turn, was washed three times with the stop solution. The amount of [3sS] Met-tRNAi which was bound in the ternary complex and retained on the filter was estimated in a scintillation counter (efficiency: 96%), with corrections made for zero-time controls. Specific activity was expressed as cpm of bound [aSS]Met-tRNAgmg of protein. Into two tubes were placed 0.2 ml of the reaction mixture for bEF-1 which contained 50 mM Tris-HCl (pH 7.4), 50 mM NH4C1, lO mM magnesium acetate, 3 mM phosphoenolpyruvate, lO pg pyruvate kynase, 3 mM [3H]GTP (approx. 0.5/~Ci; New England Nuclear, l0 Ci/mmol), and about 200/~g of the dialyzed ammonium sulfate fractions (see ref. 2). After incubation at 37°C for 5 min, the reaction in the first tube [2] was terminated with 3 ml of ice-cold 10 mM Tris-HC1 (pH 7.4), l0 mM NH4CI, and l0 mM magnesium acetate and the mixture was passed through a nitrocellulose filter (0.45 ~umpore size). The radioactivity retained on the filter was counted with 48% efficiency. Phe.tRNA 0 5 0 pmol) was added to the second tube [2] and the mixture was placed on ice for 2 rain. The mixture was then filtered and the filter was counted as described above. The difference in the radioactivity on the first and second filters, after correction for a zerotime control, represented the amount of [3H] GTP specifically bound in the ternary complex. Specific activity was expressed as cpm of bound [3H] GTP/mg protein. The controls for activity of bIF-2 in the bEF-1 reaction mixtures and vice versa gave negligible cross-measurements which were taken to be negative [7]. RESULTS Since the rate of total protein synthesis declines with age in spite of a gain in total brain mass, we measured the specific activities of the two known rate-limiting catalysts of protein synthesis at the translational level [2,8] and the cumulative brain weight during aging. These three parameters, the activities of bIF-2 and bEF-1 and brain weight, were examined in two rodent species, the rat and the mouse, which have different longevity. The intervals of time at which the measurements were made were shorter at around the beginning of postnatal life than those made later because the changes in these parameters are greater during the early postnatal period [7]. It was observed (Fig. 1)

204 that activities of both factors decreased in parallel: very rapidly during the first 2 weeks and then reaching apparently constant minimal values by day 180-270 and 6 0 - 9 0 for rat and mouse, respectively (Fig. 1A and B). The brain weight of both rat and mouse increased, reaching maximal values at about the same time as that when the activities of the translation factors remained almost constant. In agreement with published data [25,26], the brain growth for these two rodents during the first 30 days of postnatal life showed two to three periods of slow growth. This was more clearly shown here in the mouse (Fig. 1B). Although the complete life spans of these animals were not covered, the period of examination in this study was sufficiently ample to document the loss of brain mass which occurs later in life, at ~ 2 7 0 - 3 6 0 days in the rat and at ~ 1 8 0 - 2 7 0 days in the mouse. Determinations of total protein in the 150 000 g supernatant of the tissue homogenate gave a curve similar to that from brain weight determinations but with a wide variability (data not shown). Overall, each of the three curves in the rat system was similar to the corresponding curve in the mouse system (Fig. 1), with changes occurring earlier in the mouse, the species with shorter longevity. For both systems, from the gross mirror image symmetry of the curves, it appeared that there existed an inverse relationship between rate-limiting protein synthetic activities and total tissue weight. To examine both this negative correlation and the variation of the three curves in relation to time, we attempted to adjust an exponential model to the points by a semilogarithmic plotting which is shown in Fig. 2. (Note that the blF-2 and bEF-1 curves were displaced to the right for legibility.) None of the three curves in either system could be explained by a single exponential process; each required at least three components with different slopes. In each species, the first component of each curve covered about the same postnatal period (10-15 days); within a species, the absolute values o f the slopes were very similar and also most evident in the mouse (Fig. 2B): blF-2 ( [ - 0 . 0 4 1 ] ) , bEF-1 ([---0.047]), and brain weight ([0.064]). The second and third components of the curves in each species also showed similar values but the variation was greater in time as well as in the values of the slopes. Thus, the inverse correlation of the activities of the factors and tissue weight held (except during the period where brain mass diminishes), and the points in each curve required at least three different sets of parameters for the exponential model (the significance of the six sets of lines was at a level of P _<0.05, except for the third components of rat blF-2 and mouse bEF-1). To further examine this negative correlation, we analyzed the kinetics of the decay in activity in relation to brain weight gain, that is, the differential rate of decay. For this, two types of graphs may be constructed, either: (a) the activity values plotted against the increments in weight gain; or (b) the ratio of the decrements in activity to the increments in weight plotted against time. In neither method did our data produce a straight line, thereby indicating that the relationship was neither continuous, nor constant (data not shown). We then analyzed the kinetics of decay in activity. The data of Fig. 2 showed that the rate of decay was not constant, but changed at least three times during the period of examination: before puberty, during puberty and early adulthood, and thereafter. There-

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Fig. 4. Linear correlations (log/log) between the activities of blF-2 or bEF-1 and postnatal age in the rat (A) and mouse (B). The data points for mouse bEF-1 have been displaced 0.6 units to the right for clarity. The regression lines (numbered from left to right), of the general form y = bx m, were obtained by the method of least squares (on the logarithmic transformation) with the following b, m, r parameters, and P values: rat bEF-1 Yl, 3.688, --0.08, --0.948, 0.01; y~, 3.9, --0.378, -0.993, <0.001 ;)'3, 3.618, -0.11, -0.76, >0.1; y,, 4.07, -0.424, -0.992, <0.001: y~, 3.2, -0.044, 0.971,

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207 (P < 0.001, see legend of Fig. 4). The average total slope ((slope blF-2 + slope bEF-1)/2) for rat and for mouse was --0.29 and ---0.35, respectively• The ratio of slope decay (mouse/rat) was 1.2 which is very similar to the square root of the ratios (rat/mouse) obtained for approximate life span, 1.29 (x/2.5/1.5), and puberty, 1.26 (x/8/5), between our colonies of these two species. DI SCUSSION We have previously shown that the decrement in the rate of brain protein synthesis observed in cell-free systems during the postnatal period is dependent on the rates of activities of blF-2 and bEF-1, which function as rate-limiting catalysts in translation and which become qualitatively different with age. Moreover, the decay of activities of these factors correlates in time and in extent with the in vitro rates of brain protein synthesis. Since, throughout our work, we have been utilizing partially purified factors, care was taken to include experiments which could answer the obvious and appropriate criticisms related to changes in specific activities or in sensitivity to heat or chemicals by differing amounts of protein, proteases, or enzymatic effectors in the preparations. We, therefore, consider the data valid [2,7,8]. The present results on both the parallelism of decay and of pattern of decay between blF-2 and bEF-1 activities, more strongly support our previous idea [7] that a coordinated regulation of these two factors probably exists. The decay in biosynthetic activity and the simultaneous gain in cumulative brain weight had always been puzzling and led us to analyze their probable interrelations. The data on the changes of these two processes in relation with time (Figs. 1 and 2) indicated that their velocity curves are inversely related, mainly during the period of most rapid change. More detailed analysis (see Results) did not permit further clarification. However, when we compared the acceleration data, a very close mirror image symmetry was found (Fig. 3, insets). An important finding was that neither process showed smooth diminution in acceleration. Instead, each presented bursts in their respective positive or negative values followed by periods of intense recovery as if, in the processes of brain growth and biosynthetic activity, there was an antagonistic character which, in turn, may result from the possible existence of excitatory and inhibitory processes similar to those commonly

0.001; Ytot~, 3.749, --0.254, -0.977, <0.001; rat blF-2 Yl, 5.93, -0.087, -0.98, 0.01; Y2, 6.28, --0.64, -0.99, <0.001; y~, 5.6, -0.069, -0.99, 0.01; Y4, 6.28, -0.478, -0.99, <0.001; Y0, 5.07, 0.013, 0.44, >0.1; Ytatai, 5.97, -0.329, -0.986, <0.001 ; mouse bEF-1 Yt, 4.18, -0.184, -0.99, 0.001 ; Y2, 4.56, -0.82, -0.98, <0.001 ; Ya, 3.77, -0.11, -0.81,0.02; Y4, 4.29, -0.41, -0.99, 0.01 ;yj, 3.78, -0.14, -0.77, >0.1 ; Ytotal, 4.1, -0.32, -0.95, <0.001 ; mouse blF-2 Yl, 6.07, -0.27, -0.99, <0.001; Y2, 6.37, -0.75, -0.99, <0.001 ; y~, 5.64, -0.18, -0.99, 0.05; Y4, 5.75, -0.25, -0.98, 0.02;y s, 5.49, -0.12, -0.99, <0.001; Ytota, 6.02, -0.38, -0.96, <0.001, respectively.Insets: yrm= 0.33 + 0.017x (r = 0 . 0 4 8 ) ; Y m o u N = 0 . 4 6 - - 0 . 1 l x (r = - 0 . 2 1 7 ) .

208 involved in the electrical firing of neurons. From the acceleration data, it was easy to reconcile the diminution in biosynthetic activity from birth onwards despite brain weight gain, since the overall acceleration of which was also diminishing, as has been reported for human growth before and after birth [27]. It is also interesting to note that some of the spurts of acceleration in tissue weight seemed to appear somewhat later than those in the activities of the factors (more clearly seen in the mouse data, inset of Fig. 3B). The fact that a straight line could not be constructed from the ratio of the deltas of the activities of the factors to those of tissue weight plotted against time, in addition to the indication of lack of continuity and constancy of the relationship between activities and weight. would argue against a dilution of the factors by the increase in total mass during the first 100 days (Fig. 1). Within the period of present examination, brain mass loss (which presented somewhat abruptly in time) amounted to about 25% and 10% of peak adult mass in rat and mouse, respectively. These figures are similar to those (7-8%) reported for elderly human brain in postmortem studies [28]. In the human, there is a loss of gray and white matter; the ratio of gray to white matter varies from 1.28 at age 20, to 1.13 at 50 years, and to 1.55 at 100 years [29,30]. It would be of interest to know if this suggestive differential loss, and also regional differential loss, could be correlated to a differential decay in the activities of the factors. The data also showed that the previous assumption [2] that the decay in the activities of the factors during aging followed a continuous pattern was incorrect. There were definite breaks in the plots of velocity in the graphs of both the semilogarithmic and the log/log values. Furthermore, the breaks observed in the logarithmic plots corresponded somewhat not only to the breaks in tlae plots of the ratios of deltas of the activities of factors to those of time against time (Fig. 4, insets) but also to the spurts of negative acceleration in the activities of factors (Fig. 3). The possible significance of this multirate pattern to the whole process of aging is intriguing: it may suggest that organismal aging may also show different velocities during time. The overall average logarithmic curves (Fig. 4, legend) gave slope ratios (mouse to rat) very similar to the square roots of the ratios of life span and puberty time of these animals. This is an important finding since there is strong evidence that the life spans of fibroblasts (measured as population doublings in vitro), erythrocytes (as circulating cells in vivo), and perhaps lymphocytes (as survival in vitro) from several mammals are related to the life spans of the corresponding species by this power law [31]. The similarity of values reported herein would then indicate that the velocity of decay of brain protein synthesis factors: (a) is related to the life spans of cells and organisms, thus linking these three levels of organization;and (b) is within a putative common biological process of aging, that may be similar to the genetic process in which the ced-3 and ced-4 functions are involved in the programmed cell death during neurogenesis in a nematode [32]. Also, since the present f'mdings are independent of accumulation with age of altered proteins and/or changes occurring in mid- or late life, they add to the argument against aging as being a stocastic process and a postgrowth and postmaturational phenomenon [7].

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