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Rate of RNA turnover in rat liver in relation to intake of protein
ARCHIVES
OF
BIOCHEMISTRY
Rate
AND
of RNA
CYRIL Physiological
Chemistry
BIOPHYSICS
138,
%%k%9
Turnover
in Rat
to Intake
of
0. ENWONWU’
Laboratories,
Department
of Technology,
Received
(1970)
December
AND
Liver
in Relation
Protein HAMISH
N. MUNRO
of Nutrition and Food Cambridge, Massachusetts 2, 1969;
accepted
March
Science,
Massachusetts
Institute
13, 1970
The effect of dietary intake of protein on the turnover of liver RNA has been measured in rats by observing the rate of loss of label from liver ribosomal RNA over a period of several days after injection of aH-labeled erotic acid. Rats receiving a normal intake of protein showed fractional rates of RNA synthesis and degradation of about 2045% daily. When rats were fed a diet free of protein, RNA breakdown was at first accelerated to over 40% per day. However, after 2 days on the protein-free diet the fractional rate of degradation fell to about 15$& daily, and the rate of RNA synthesis to an even lower level, so that animals on this diet showed a continuous reduction in the RNA content of their livers. On the basis of the present and earlier studies, it is suggested that RNA turnover is regulated through changes in the population of free ribosomal subunits in the liver cytoplasm, the abundance of subunits being affected by amino acid supply.
The amount of RNA in the liver is subject to nutritional control. When a rat is starved (1, 2) or is fed on a protein-deficient diet (3, 4), there is an immediate and extensive loss of RNA from the liver for about 2 days, after which a new and lower plateau is attained. On the basis of incorporation studies employing 14C-glycine as a purine precursor, Clark et al. (5) concluded that the rapid reduction in liver RNA content during the first 24-48 hr on a protein-deficient diet is due to accelerated breakdown of liver RNA rather than to reduction in rate of RNA synthesis. These experiments and subsequent studies (6) have led us to the conclusion (7) that the RNA content of the liver is determined at least in part by changes in rate of RNA breakdown caused by variations in amino acid supply. The objective of the present studies has been to test this interpretation by direct measurement of the rate of RNA turnover in the livers of rats receiving adequate or low intakes of pror Present address: (B-122), University Washington 98105.
Health Sciences of Washington,
Building Seattle, 532
tein, including the transition period between one level of protein intake and another. The rate of degradation of RNA in the livers of rats receiving a normal diet has been measured by several authors. In 1960, Gerber et al. (8) examined the rate of loss of label from both the ribose and the bases of liver RNA and concluded that the mean turnover time of RNA in the liver of the rat is 7.2 days; this is equivalent to a halflife of about 5 days. This figure for total liver RNA agreeswith later estimates of the half-life of liver ribosomal RNA (!&la), with the exception of one low figure of 40 hr (13). Only one study of factors affecting RNA turnover appears to have been made. Hirsch and Hiatt (10) examined the effect of total starvation on the turnover of rat liver RNA and concluded that the loss of RNA that occurs during starvation is due to an increase in ribosomal RNA degradation, as well as to reduction in rate of RNA sythesis. Starvation represents total withdrawal of nutrients, so that it is not possible to evaluate the role of individual nutrients in the observed response. In our studies of
LIVER
RNA
TURNOVER
liver RNA turnover, intake of protein was the only variable. The results show that protein intake by itself does affect the rate of turnover of RNA in the liver. MATERIALS
AND
METHODS
Animals a,nd diets. Male rats (Charles River, Mass.) weighing 20&250 g were housed individually in an air-conditioned room with lighting regulated. During the experiments they were fed on synthetic diets prepared in the form of agar gels (14). The normal (protein-containing) diet consisted of l@T, casein supplemented with 0.3% methionine, 20.7% glucose, 16.7% sucrose, 20.4yo dextrin, 15%) cottonseed oil (Wesson Oil Co., Fullerton, California), 4% salt mixture (General Biochemicals, Chagrin Falls, Ohio), 1% vitamin mixture (14), 0.2yo choline chloride, and 3.5% agar. To prepare a protein-free diet, the casein and methionine were omitted, and the amounts of glucose, sucrose, and dextrin were increased to replace these constituents. To prepare the diet, the agar was dissolved in hot water and the dry diet constituents were mixed into it; the vitamin mixture and choline were added when the agar solution was cooler. After setting, the agar gel diets were cut into blocks for feeding. Isotopic labeling of liver RNA. The rats were injected intraperitoneally with erotic acid-5-3H dissolved in 0.97& NaCl. The dose varied from 20-50 PCi in different experiments, as indicated. The isotope was administered either after the rats had been stabilized for 3 days on the adequate or lowprotein diets (Expt. Series 1) or else before the animals had been separated into adequate and low-protein dietary groups (Expt. Series 2). Tissue fractionation. Groups of rats were killed by guillotine (Harvard Apparatus Co., Dover, Massachusetts). The livers were quickly chilled in several volumes of ice-cold 0.375 M sucrose made up in TKM buffer (0.05 NI Tris, pH 7.6; 0.02 M KCl, 0.005 M MgCh), weighed, briefly minced with scissors, and homogenized in 2 vol of the same medium, using a chilled Potter-Elvehjem homogenizer with a close-fitting Teflon pestle. Cell debris, nuclei, and mitochondria were then sedimented by centrifugation at 15,OoOg for 10 min in Rotor 30 of the Spinco L2 ultracentrifuge. The supernatant fluid was treated with 1% deoxycholate to dissolve membranes and then was used to prepare polyribosome patt.erns and to recover the ribosomal RNA for study of isotope incorporation. Liver homogenates used for polysome profile preparations were initially treated with antiferritin serum in order to exclude ultraviolet absorption due to ferritin (15). The polysome patterns were obtained by applying the deoxycholate-
AND
PROTEIN
INTAKE
533
treated
post-mitochondrial supernatant fraction to a l(r40$& continuous sucrose gradient prepared according to a modification (16) of the original method of Bock and Ling (17). The areas under the monosome, disome, and polysome peaks were measured by planimeter (6). For purposes of calculation, the monosome and disome areas were combined since Reader and Stanners (18) has shown that disomes occur largely artifactually by monosome aggregation during preparation. In order to measure the amount of erotic acidJH incorporated by the liver ribosomal RNA, the deoxycholate-treated post-mitochondrial fraction was prepared according to the method of Hirsch and Hiatt (10). The ribosomes were isolated by centrifuging the fraction as described by these authors and were then solubilized in NCS reagent and counted; radioactivity was measured with a Packard liquid scintillation counter using Bray’s scintillation solution (19). The biological half-life of ribosomal RNA was obtained by plotting the specific activity of the RNA against time after isotope injection on semilogarithmic graph paper. The formula t+/ln 2 provides the turnover time, and from this the percentage of the RNA pool replaced daily can be derived from the formula 100 In 2/ti . Nucleic acid content of liver. Liver RNA content was obtained by the modified Schmidt-Thannhauser method of Fleck and Munro (20). DNA was measured by Ceriotti’s method using 2.5 N HCI in place of concentrated HCI (21) and prolonging the heating time to 20 min instead of 10 min (21). The standard was calf thymus DNA.
directly
RESULTS
First series of experiments. The objective in these experiments was to examine the turnover of liver RNA in rats habituated to either the synthetic 18% protein diet or a similar diet in which the protein was replaced by carbohydrate. Since both diets were fed in similar amounts, the only variable was the presence or absence of dietary protein. In the first of these experiments, two groups of rats, each with a mean weight of 203 g, were fed on 15 g/day of either the 18 % protein diet or the protein-free diet. After a period of 3 days in order to accustom them to these diets, both groups received an intraperitoneal injection of 3H-erotic acid. Three animals from each dietary group were killed after 3, 5, 7, or 10 days on the diets and their livers were analyzed for total RNA and DNA, and for the specific activity of
534
ENWONWU
AND TABLE
EFFECTOFPROTEININTAKEONAMOUNTOF FROM Days
Animals 3 5 7 10 Animals 3 5 7 10
on 18%
after
protein
on Protein-free
Mean
‘H
wt.
RNA LIVER
MUNRO I
INRATLIVERANDONRATEOF RIBOSOMAL RNA
liver kJ
Liver
RNA
content
Loss
OFRADIOACTIVITY
3H activity cpm/mg
RNA
of ribosomal
RNA
Mdg
Mg/liver
cpdliver
8.4 7.4 9.2 8.9
6.5 7.4 6.3 6.7
54.9 54.0 57.7 59.9
2,830 1,880 1,350 435
156,000 97,000 78,000 26,000
8.1 6.6 7.2 6.6
5.1 5.0 4.8 4.8
41.1 32.9 34.8 31.5
2,180 1,890 1,570 830
90,ooo 62,000 55,000 26,000
diet
diet
ribosomal RNA. The basic data are shown in Table I. Since the animals were nearly identical in initial body weight, the RNA and DNA content of the livers of the two groups could be directly compared by expressing them as the total amount per rat liver. It was found in all experiments that the short periods on the various diets had no significant effect on the amount of DNA in the liver and the data for DNA are, therefore, not reported. In order to measure ribosomal RNA turnover, the specific activity of RNA in the ribosomes isolated from each liver was measured and then multiplied by the total RNA content of the same liver, thus providing the total amount of radioactivity remaining in the liver ribosomal RNA. The use of the total liver RNA in place of total ribosomal RNA for this calculation is dictated by the difficulty of isolating ribosomal RNA quantitatively and by the observation of Hirsch (10, 22) that ribosomal RNA provides about 90 % of the total RNA in the liver under various nutritional conditions. The daily fractional change in amount of RNA and its fractional loss of radioactivity were obtained by plotting the data on a logarithmic scale against time in days on each diet (Fig. 1). Figure 1 shows that the total amount of RNA contained in the livers of rats receiving a fixed amount (15 g) of the 18 % casein diet rose slightly (+ 1% per day) during the period between days 6 and 13 on the diet, whereas the animals on a similar intake of
the protein-free diet showed a daily fractional RNA loss of -3 % per day. Figure 1 also shows the loss of radioactivity from total liver RNA from Day 3 to Day 10 after labeled erotic acid injection (Days 6-13 on the diets). It has been observed by Hirsch and Hiatt (10) and confirmed by us that incorporation from precursors ceases to increase by the second day after injection and thereafter falls off exponentially. Figure 1 demonstrates that thereafter the plot of log total radioactivity in ribosomal RNA is linearly related to number of days after injection, confirming the relationship established by Hirsch and Hiatt (10) for the specific activity of liver ribosomal RNA. From the linear plots, it can be calculated that the half-life of ribosomal RNA is 65 hr on the 18% casein diet, equivalent to a turnover time of 94 hr, and thus, a fractional degradation rate of 26% of liver RNA per day. If the liver of these animals neither gained nor lost RNA, then synthesis would also be at the fractional rate of 26% per day. Since in this experiment the total RNA content on this diet gained at the rate of 1% per day, the fractional rate of synthesis must be higher than the rate of degradation by this amount, and thus is 27 % of liver RNA per day. In the case of the animals on the protein-free diet, Fig. 1 shows that degradation was slower, with a half-life of 96 hr, equivalent to a turnover time of 139 hr and a fractional degradation rate of 17% per day. Since the rats lost 3% of their liver
LIVER
RNA
TURNOVER
AND
PROTEIN
535
INTAKE TABLE
II
EFFECT OF PROTEIN INTAKE ON THE DAILY FRACTIONSL RATE OF CHANGE IN RAT LIVER RNA CONTENT, AND ITS RATES OF DEGRADATION
AND
SYNTHESIS 1 Daily
Expt’l series
IA
IB
II
Dieta
18% 0% 18% 0% 18yc 0% 0%
L
-!-r L Days
after
H3
- orot~c
ocld
FIG. 1. Total amount of RNA per liver and rate of loss of 3H-RNA from the livers of rats receiving a diet adequate in protein (O---O) and of rats adapted for several days to a protein-free diet (o-----O). The rats were fed equal amounts of either a diet containing 18% protein or a proteinfree diet. On the third day they were injected with erotic acid-3B. and measurements of total RNA and 3H-RNA per liver were made from 3 days after isotope administration onward. The amounts of total RNA and of 3H-RNA per liver are plotted on a logarithmic scale. Each point represents results from the pooled livers of three animals.
RNA per day, the fractional synthetic rate must have been less than that for degradation, namely, 14% per day. These estimates of rates of degradation and synthesis are set out in Table II, which also includes a replicate experiment in which only 10 g of each diet was fed per day. Essentially the same picture was obtained, namely, that the turnover of liver RNA slows down in protein deficiency, there being both retarded synthesis and retarded breakdown. The latter exceeds synthesis, so that a persistent slow loss of RNA occurs as a result of lack of protein in the diet. Second series of experiments. The objective here was to examine liver RNA turnover during the transition period from an ade-
Protein Protein (from days on) Protein Protein (from days on) Protein Protein (first days) Protein (from days on)
3
fractional
rate
(%)
Change in RNA
;y.g a rate
$1 - 3
-26 -17
+27
0 6
-23 -12
$23 +6
-21 -43
+22
-15
+10
3
-
2
+1 -10
3
-
5
(1 Fifteen grams of each diet were IA and II, and 10 g. in Expt. IB.
.-
Spthetic rate
+14
f33
fed in Expts.
quate protein intake to a protein-free diet, since previous studies (3, 4) showed that liver RNA content undergoes a considerable reduction at this time, and there was some evidence to suggest that this occurred because of accelerated RNA breakdown. Twenty-one rats of initial weight close to 230 g (range 230-237 g) were fed the 18% casein diet ad libitum for 3 days, and were then injected with 3H-orotic acid. The 18% casein diet was then fed for a further 2 days at a level of 15 g per day, after which three rats were killed to provide an initial level for the quantity of RNA in each liver and its isotope content at this point. The remaining rats were divided into two groups of equal average weight. One group continued to receive 15 g daily of the 18% casein diet, while the other group was started on 15 g daily of the protein-free diet. Groups of three rats from each diet were killed on the second, fourth, and seventh days after separating the animals into two groups. Figure 2 shows the changes produced in total liver RNA and in radioactivity retained in total ribosomal RNA, and the fractional rates of change computed from these data are summarized in Table II. The total RNA content of the livers of rats receiving the 18% pro-
536
ENWONWU
‘\
6 t
Days
afler
3H
-erotic
acid
FIG. 2. Total amount of RNA per liver and rate of loss of 3H-RNA from the livers of rats receiving a diet adequate in protein (@---a) and of rats immediately after changing to a protein-free diet (O-----O). All animals were fed the diet containing adequate amounts of protein until 2 days after erotic acid-3H administration; one group was then given a similar amount of a protein-free diet. The amounts of total RNA and of 3H-RNA per liver are plotted on a logarithmic scale. Each point represents results from the pooled livers of three animals.
tein diet rose slightly (+ 1% per day) during the experimental period. In agreement with the findings of previous investigators (3, 4), withdrawal of protein resulted in a loss of liver RNA that occurred in two phases. During the first 2 days, there was a sharp reduction at a fractional rate of 10% per day. Thereafter, the rate of loss slowed to some 5 % per day, similar to that found in the two experiments reported above, in both of which the rats were allowed to adjust for a few days to the low intake of protein before measurements of liver RNA content were made. The loss of radioactivity by the two groups of rats is also shown in Fig. 2. Rats on the 18% casein diet throughout the experiment showed a progressive decrease in total radioactivity in the liver RNA with a half-life of
AND
MUNRO
81 hr. Consequently, the fractional rate of degradation was 21% and the rate of synthesis was 22%, being thus similar to the turnover rates found on this diet in the other experiments shown in Table II. The rats that received the protein-free diet showed extensive changes in rate of turnover of RNA. During the first 2 days after deleting protein from the diet, degradation was considerably accelerated, and at all times thereafter the rats on the protein-free diet had less radioactive RNA in their livers than remained in the livers of animals receiving adequate amounts of dietary protein for a similar length of time (Fig. 2). During the first 2 days after withdrawal of dietary protein, the half-life of ribosomal RNA was computed to be 38 hr from the slope between Days 2 and 4 after 3H-orotic acid administration. Consequently, the fractional rate of RNA breakdown was 43 % per day, which is twice as fast as the rate of degradation on the 18% casein diet. Since the loss of RNA during this 2 day period was only 10% per day, the fractional rate of synthesis can be computed to be 33% per day. These estimates of turnover do not have a high degree of precision since they are based on only two points instead of the three or more points used for calculation of turnover for animals equilibrated in the diets. Nevertheless, it can be concluded that the immediate loss of liver RNA after deprivation of dietary protein is due to a sudden acceleration in rate of RNA breakdown. On continuing to feed the protein-free diet, degradation of RNA quickly slowed down to somewhat less than that observed for rats on the 18% casein diet, and the fractional rate of RNA synthesis also became even slower. These latter findings for rats conditioned to the protein-free diet agree with those observed in the earlier experiments recorded in Table II. Ribosome aggregation and protein intake. Since our studies showed a reduction in ribosomal RNA breakdown in the livers of animals receiving a protein-free diet for several days, it was of interest to determine the sizes of ribosome aggregates in the livers of these rats as compared with animals receiving an adequate intake of protein.
LIVER RNA TURNOVER 0.5 NORMAL .----
MALE
RATS
0% CASEIN 16 % CA~EIN
FOR 9 DAYS (IOg/RAT/DAY) FOR 9 DAYS (KI~/RAT/DAY)
i 0.4
t-
AND
PROTEIN
INTAKE
537
monosomes and disomes in the liver and the total concentration per g of liver is much lower in the case of the protein-deficient animals, due to extensive loss of RNA by this group. Evidence obtained from an earlier investigation (23, 24) shows that ribosome subunits are much fewer in the livers of rats that have received a proteinfree diet for several days. This infrequency of monosomes and subunits is of significance when we come to consider the mechanism regulating RNA breakdown. DISCUSSION
1 10% -40% TOP
SUCROSE
DENSITY
GRADIENT
1 BOTTOM
FIG. 3. Polysome profiles on sucrose gradients obtained from the livers of rats fed equal amount of either an 18% protein diet (----) or a proteinfree diet (--) for 9 days. The livers of four rats on each diet were combined and amounts of cytoplasmic extract equivalent to the same weight of liver from ea,ch group were put on each sucrose gradient.
of rats were fed 10 g of either the 18% protein diet or the protein-free diet for 11 days, and were killed on the morning of Day 12. The livers from individuals in each dietary group were pooled and analyzed. Those fed on the 18 % casein diet had 44 mg RNA per liver, whereas those on the proteinfree diet had 20 mg RNA/liver. Figure 3 shows the total polysome profiles obtained from the livers of animals on each diet. The smaller profile on the protein-free diet is due to the fact that polysomes prepared from equal weights of liver were layered onto each gradient, and the concentration of RNA per g of liver was less in the case of the rats on the protein-free diet (cf. Table I). The percentages of monosomes plus disomes in the two profiles were very little different in the two profiles (39% on the proteincontaining diet and 44 % on the protein-free diet); in consequence, the total number of Groups
The general characteristics of ribosomal RNA turnover in the liver have been examined by Hirsch and Hiatt (10). They examined the rate of loss of label from liver ribosomes after injection of both Wguanido-arginine to label ribosome protein and 3H-erotic acid to label RNA. The guanido group of free arginine is so quickly exchanged in the arginineeornithine cycle that the label is rapidly obliterated when guanido-labeled arginine is released from liver proteins. Consequently, this radioactive amino acid is used to obtain true halflives of liver proteins without significant recycling of the label (25). In Hirsch and Hiatt’s study, ribosomal protein and ribosomal RNA gave identical half-lives of 5 days; this indicates that the pyrimidine bases labeled from 3H-erotic acid are not significantly recycled, and, consequently, that this label provides a good approximation to the true rate of degradation of ribosomal RNA. Hirsch and Hiatt (10) also examined the effect of prolonged starvation on the rates of RNA degradation and synthesis, using changes in quantity of RNA per unit of DNA as well as loss of specific activity to make these calculations. They observed that the fractional rate of RNA degradation increased and the fractional rate of RNA synthesis decreased during starvation. This resulted in a rapid and extensive loss of RNA per cell, accompanied by a loss of lo-25 % of liver DNA, which would be indicative of some cell turnover. This large change in the amounts of DNA per liver due to starvation is contrary to the findings of others (1, 2) who reported no change in DNA over a
53s
ENWONWU
2-day period of starvation. In a recent paper (26), Hirsch et al. reported that their procedure for measuring liver DNA gives invalid results in starving animals. Consequently, the values for RNA turnover in starving animals calculated by Hirsch and Hiatt (10) may also require revision. Starvation involves withdrawal of all nutrients. In our present series of experiments, we have varied only intake of protein, since earlier studies (5, 6) had suggested that availability of amino acids affects rate of degradation of liver RNA. The present studies show that animals habituated to a diet deficient in protein undergo a reduction in rate of both synthesis and degradation of ribosomal RNA. The latter response is contrary to the effect of starvation observed by Hirsch and Hiatt (10). The diminished rate of RNA synthesis found in our proteindepleted animals contrasts with the increase in RNA polymerase activity observed in the liver nuclei of protein-depleted rats by Shaw and Fillios (27). No explanation can be offered at present for this discrepancy. The transition from a diet adequate in protein content to one deficient in protein is of special interest since there is an initial rapid loss of RNA from the liver at the time of change. The immediate effect of withdrawing protein from the diet is not a slowing down of degradation but a sudden acceleration for the first 2 days, followed by retarded degradation from then on. The immediate increase in degradation agrees with our earlier studies (4, 5) in which incorporation of 32Pand of 14C-glycine into the purines of liver RNA and its precursors was examined. During the first day of protein withdrawal, incorporation of 14C-glycine into RNA was greatly reduced, and this was shown to reflect similar labeling changes in the precursor pools of adenine and guanine compounds. The effect was attributed to filling of the precursor pools with breakdown products of RNA degradation (5) ; possibly this caused feedback inhibition of purine biosynthesis. As soon as this short, initial period of intense RNA degradation is over, the uptake of precursor into purines resumes again (5). Changes in acid-soluble purine labeling were also observed a few hours after
AND
MUNRO
a tryptophan-deficient mixture of amino acids was fed to rats in place of a complete amino acid mixture (as), and it was subsequently shown (6, 29) that the absence of tryptophan from the mixture causes an accumulation of monosomes and free ribosome subunits in the liver cell. Since we have shown that the free ribosome subunits are rich in endogenous ribonuclease (23), it was concluded (7) that tryptophan deficiency or general protein deficiency results in accumulation of subunits and thus in accelerated RNA breakdown, which proceeds until a new equilibrium between polysomes, monosomes, and subunits is established. This occurs after a few days of protein depletion, as shown by the absence of excess monosomes in the polysome profiles of protein-depleted animals (Fig. 3). On the basis of this hypothesis, the fractional rate of RNA degradation will be dependent on the subunit population of the cell. In fact, Munro et al. (24) found that the subunit fraction of the cell was most abundant during the period of rapid RNA loss during starvation, less for animals during absorption of a protein meal, and least after a few days of a protein-free diet. This change in subunit abundance thus correlates well with changes in the rate of loss of label from ribosomal RNA (Table II). It is of interest that the rate of RNA synthesis does not fall as soon as the protein-free diet is given, but only after extensive RNA loss has occurred (Table II). The nature of this control over synthetic rate is not known. REFERENCES 1. DAVIDSON, 2.
3. 4.
5. 6. 7. 8.
J. N., Cold Spring Harbor Symp. @ant. BioE. 12, 50 (1947). THOMSON, R. Y., HEAGY, R. C., HUTCHISON, w. c., AND DAVIDSON, J. N., Biochem. J. 63,460 (1953). KOSTERLITZ, H. W., J. Physiol. 106,194 (1947). MUNRO, H. N., NAISMITH, D. J., AND WIKRAMANAYAKE, T. W., Biochem. J. 64, 198 (1953). CLARK, C. M., NAISMITH, D. J., AND MUNRO, H. N., Biochim. Biophys. Acta 23,587 (1957). WUNNER, W. H., BELL, J., AND MUNRO, H. N., Biochem. J. 101, 417 (1966). MUNRO, H. N., Fed. Proc. 2’7, 1231 (1968). GERBER, G., GERBER, G., AND ALTMAN, K. I., J. Biol. Chem. a36, 2682 (1960).
LIVER
RNA
TURNOVER
9. LOEB, J. N., HOWELL, R. R., AND TOMKINS, G. M., Science 149, 1093 (1965). 10. HIRSCH, C. A., AND HIATT, H. H., J. Biol. Chem. 241,5936 (1966). 11. WILSON, S. H., AND HObGLAND, M. B., Bio&em. 1. 103, 556 (1967). 12. BLOBEL, G., AND POTTER, V. R., Biochim. Biophys. Acta 166, 48 (1968). 13. HADJIOLCIV, A. A., Biochim. Biophys. Acta 119, 547 (1966). 14. ROGERS, Q. R., AND HARPER, A. E., J. Nutr. 87, 267 (1965). 15. DRYSDALW, J. W., AND MUNRO, H. N., Biochim. Biophys. Acta 138, 616 (1967). 16. BRITTEN, R. J., AND ROBERTS, R. B., Science 131, 32 (1960). 17. BOCK, R. M., L4~~ LING, N. S., Anal. Chem. 26,1543 (1954). 18. READER, .R. W., AND STANNERS, C. P., J. Mol. Biol. 28, 211 (1967). 19. BRAY, G. A., Anal. Biochem. 1, 279 (1960).
AND 20.
PROTEIN MUNRO,
539
INTAKE H. N.,
AND
FLECK,
A., Analyst
91,78
(1966). 21. KECK, K., Arch. Riochem. Biophys. 63, 446 (1956). 22. HIRSCH, C. A., J. Biol. Chem. 242,2822 (1967). 23. HIRD, H. J., MCLEAN, E. J. T., BND MUNRO, H. N., Biochim. Biophys. Acta 87,219 (1964). 24. MUNRO, H. N., MCLEAN, E. J. T., AND HIRD, H. J., J. Nutr. 83, 186 (1964). 25. SWICK, R. W., J. Biol. Chem. 231, 751 (1958). 26. EDELMAN, M., HIRSCH, C. A., HIATT, H. H., AND Fox, M., Biochim. Biophys. Acta 179, 172 (1969). 27. SHAW, C., AND FILLIOS, L., J. Nutr. 96, 327 (1968). 28. MUNRO, H. N., AND CLARK, C. M., Biochim. Biophys. Acta 33, 551 (1959). 29. PRONCZUK, A. W., BALIGA, B. S., TRIANT, J., AND MUNRO, H. N., Biochim. Biophys. Acta 167, 204 (1968).
OF
BIOCHEMISTRY
Rate
AND
of RNA
CYRIL Physiological
Chemistry
BIOPHYSICS
138,
%%k%9
Turnover
in Rat
to Intake
of
0. ENWONWU’
Laboratories,
Department
of Technology,
Received
(1970)
December
AND
Liver
in Relation
Protein HAMISH
N. MUNRO
of Nutrition and Food Cambridge, Massachusetts 2, 1969;
accepted
March
Science,
Massachusetts
Institute
13, 1970
The effect of dietary intake of protein on the turnover of liver RNA has been measured in rats by observing the rate of loss of label from liver ribosomal RNA over a period of several days after injection of aH-labeled erotic acid. Rats receiving a normal intake of protein showed fractional rates of RNA synthesis and degradation of about 2045% daily. When rats were fed a diet free of protein, RNA breakdown was at first accelerated to over 40% per day. However, after 2 days on the protein-free diet the fractional rate of degradation fell to about 15$& daily, and the rate of RNA synthesis to an even lower level, so that animals on this diet showed a continuous reduction in the RNA content of their livers. On the basis of the present and earlier studies, it is suggested that RNA turnover is regulated through changes in the population of free ribosomal subunits in the liver cytoplasm, the abundance of subunits being affected by amino acid supply.
The amount of RNA in the liver is subject to nutritional control. When a rat is starved (1, 2) or is fed on a protein-deficient diet (3, 4), there is an immediate and extensive loss of RNA from the liver for about 2 days, after which a new and lower plateau is attained. On the basis of incorporation studies employing 14C-glycine as a purine precursor, Clark et al. (5) concluded that the rapid reduction in liver RNA content during the first 24-48 hr on a protein-deficient diet is due to accelerated breakdown of liver RNA rather than to reduction in rate of RNA synthesis. These experiments and subsequent studies (6) have led us to the conclusion (7) that the RNA content of the liver is determined at least in part by changes in rate of RNA breakdown caused by variations in amino acid supply. The objective of the present studies has been to test this interpretation by direct measurement of the rate of RNA turnover in the livers of rats receiving adequate or low intakes of pror Present address: (B-122), University Washington 98105.
Health Sciences of Washington,
Building Seattle, 532
tein, including the transition period between one level of protein intake and another. The rate of degradation of RNA in the livers of rats receiving a normal diet has been measured by several authors. In 1960, Gerber et al. (8) examined the rate of loss of label from both the ribose and the bases of liver RNA and concluded that the mean turnover time of RNA in the liver of the rat is 7.2 days; this is equivalent to a halflife of about 5 days. This figure for total liver RNA agreeswith later estimates of the half-life of liver ribosomal RNA (!&la), with the exception of one low figure of 40 hr (13). Only one study of factors affecting RNA turnover appears to have been made. Hirsch and Hiatt (10) examined the effect of total starvation on the turnover of rat liver RNA and concluded that the loss of RNA that occurs during starvation is due to an increase in ribosomal RNA degradation, as well as to reduction in rate of RNA sythesis. Starvation represents total withdrawal of nutrients, so that it is not possible to evaluate the role of individual nutrients in the observed response. In our studies of
LIVER
RNA
TURNOVER
liver RNA turnover, intake of protein was the only variable. The results show that protein intake by itself does affect the rate of turnover of RNA in the liver. MATERIALS
AND
METHODS
Animals a,nd diets. Male rats (Charles River, Mass.) weighing 20&250 g were housed individually in an air-conditioned room with lighting regulated. During the experiments they were fed on synthetic diets prepared in the form of agar gels (14). The normal (protein-containing) diet consisted of l@T, casein supplemented with 0.3% methionine, 20.7% glucose, 16.7% sucrose, 20.4yo dextrin, 15%) cottonseed oil (Wesson Oil Co., Fullerton, California), 4% salt mixture (General Biochemicals, Chagrin Falls, Ohio), 1% vitamin mixture (14), 0.2yo choline chloride, and 3.5% agar. To prepare a protein-free diet, the casein and methionine were omitted, and the amounts of glucose, sucrose, and dextrin were increased to replace these constituents. To prepare the diet, the agar was dissolved in hot water and the dry diet constituents were mixed into it; the vitamin mixture and choline were added when the agar solution was cooler. After setting, the agar gel diets were cut into blocks for feeding. Isotopic labeling of liver RNA. The rats were injected intraperitoneally with erotic acid-5-3H dissolved in 0.97& NaCl. The dose varied from 20-50 PCi in different experiments, as indicated. The isotope was administered either after the rats had been stabilized for 3 days on the adequate or lowprotein diets (Expt. Series 1) or else before the animals had been separated into adequate and low-protein dietary groups (Expt. Series 2). Tissue fractionation. Groups of rats were killed by guillotine (Harvard Apparatus Co., Dover, Massachusetts). The livers were quickly chilled in several volumes of ice-cold 0.375 M sucrose made up in TKM buffer (0.05 NI Tris, pH 7.6; 0.02 M KCl, 0.005 M MgCh), weighed, briefly minced with scissors, and homogenized in 2 vol of the same medium, using a chilled Potter-Elvehjem homogenizer with a close-fitting Teflon pestle. Cell debris, nuclei, and mitochondria were then sedimented by centrifugation at 15,OoOg for 10 min in Rotor 30 of the Spinco L2 ultracentrifuge. The supernatant fluid was treated with 1% deoxycholate to dissolve membranes and then was used to prepare polyribosome patt.erns and to recover the ribosomal RNA for study of isotope incorporation. Liver homogenates used for polysome profile preparations were initially treated with antiferritin serum in order to exclude ultraviolet absorption due to ferritin (15). The polysome patterns were obtained by applying the deoxycholate-
AND
PROTEIN
INTAKE
533
treated
post-mitochondrial supernatant fraction to a l(r40$& continuous sucrose gradient prepared according to a modification (16) of the original method of Bock and Ling (17). The areas under the monosome, disome, and polysome peaks were measured by planimeter (6). For purposes of calculation, the monosome and disome areas were combined since Reader and Stanners (18) has shown that disomes occur largely artifactually by monosome aggregation during preparation. In order to measure the amount of erotic acidJH incorporated by the liver ribosomal RNA, the deoxycholate-treated post-mitochondrial fraction was prepared according to the method of Hirsch and Hiatt (10). The ribosomes were isolated by centrifuging the fraction as described by these authors and were then solubilized in NCS reagent and counted; radioactivity was measured with a Packard liquid scintillation counter using Bray’s scintillation solution (19). The biological half-life of ribosomal RNA was obtained by plotting the specific activity of the RNA against time after isotope injection on semilogarithmic graph paper. The formula t+/ln 2 provides the turnover time, and from this the percentage of the RNA pool replaced daily can be derived from the formula 100 In 2/ti . Nucleic acid content of liver. Liver RNA content was obtained by the modified Schmidt-Thannhauser method of Fleck and Munro (20). DNA was measured by Ceriotti’s method using 2.5 N HCI in place of concentrated HCI (21) and prolonging the heating time to 20 min instead of 10 min (21). The standard was calf thymus DNA.
directly
RESULTS
First series of experiments. The objective in these experiments was to examine the turnover of liver RNA in rats habituated to either the synthetic 18% protein diet or a similar diet in which the protein was replaced by carbohydrate. Since both diets were fed in similar amounts, the only variable was the presence or absence of dietary protein. In the first of these experiments, two groups of rats, each with a mean weight of 203 g, were fed on 15 g/day of either the 18 % protein diet or the protein-free diet. After a period of 3 days in order to accustom them to these diets, both groups received an intraperitoneal injection of 3H-erotic acid. Three animals from each dietary group were killed after 3, 5, 7, or 10 days on the diets and their livers were analyzed for total RNA and DNA, and for the specific activity of
534
ENWONWU
AND TABLE
EFFECTOFPROTEININTAKEONAMOUNTOF FROM Days
Animals 3 5 7 10 Animals 3 5 7 10
on 18%
after
protein
on Protein-free
Mean
‘H
wt.
RNA LIVER
MUNRO I
INRATLIVERANDONRATEOF RIBOSOMAL RNA
liver kJ
Liver
RNA
content
Loss
OFRADIOACTIVITY
3H activity cpm/mg
RNA
of ribosomal
RNA
Mdg
Mg/liver
cpdliver
8.4 7.4 9.2 8.9
6.5 7.4 6.3 6.7
54.9 54.0 57.7 59.9
2,830 1,880 1,350 435
156,000 97,000 78,000 26,000
8.1 6.6 7.2 6.6
5.1 5.0 4.8 4.8
41.1 32.9 34.8 31.5
2,180 1,890 1,570 830
90,ooo 62,000 55,000 26,000
diet
diet
ribosomal RNA. The basic data are shown in Table I. Since the animals were nearly identical in initial body weight, the RNA and DNA content of the livers of the two groups could be directly compared by expressing them as the total amount per rat liver. It was found in all experiments that the short periods on the various diets had no significant effect on the amount of DNA in the liver and the data for DNA are, therefore, not reported. In order to measure ribosomal RNA turnover, the specific activity of RNA in the ribosomes isolated from each liver was measured and then multiplied by the total RNA content of the same liver, thus providing the total amount of radioactivity remaining in the liver ribosomal RNA. The use of the total liver RNA in place of total ribosomal RNA for this calculation is dictated by the difficulty of isolating ribosomal RNA quantitatively and by the observation of Hirsch (10, 22) that ribosomal RNA provides about 90 % of the total RNA in the liver under various nutritional conditions. The daily fractional change in amount of RNA and its fractional loss of radioactivity were obtained by plotting the data on a logarithmic scale against time in days on each diet (Fig. 1). Figure 1 shows that the total amount of RNA contained in the livers of rats receiving a fixed amount (15 g) of the 18 % casein diet rose slightly (+ 1% per day) during the period between days 6 and 13 on the diet, whereas the animals on a similar intake of
the protein-free diet showed a daily fractional RNA loss of -3 % per day. Figure 1 also shows the loss of radioactivity from total liver RNA from Day 3 to Day 10 after labeled erotic acid injection (Days 6-13 on the diets). It has been observed by Hirsch and Hiatt (10) and confirmed by us that incorporation from precursors ceases to increase by the second day after injection and thereafter falls off exponentially. Figure 1 demonstrates that thereafter the plot of log total radioactivity in ribosomal RNA is linearly related to number of days after injection, confirming the relationship established by Hirsch and Hiatt (10) for the specific activity of liver ribosomal RNA. From the linear plots, it can be calculated that the half-life of ribosomal RNA is 65 hr on the 18% casein diet, equivalent to a turnover time of 94 hr, and thus, a fractional degradation rate of 26% of liver RNA per day. If the liver of these animals neither gained nor lost RNA, then synthesis would also be at the fractional rate of 26% per day. Since in this experiment the total RNA content on this diet gained at the rate of 1% per day, the fractional rate of synthesis must be higher than the rate of degradation by this amount, and thus is 27 % of liver RNA per day. In the case of the animals on the protein-free diet, Fig. 1 shows that degradation was slower, with a half-life of 96 hr, equivalent to a turnover time of 139 hr and a fractional degradation rate of 17% per day. Since the rats lost 3% of their liver
LIVER
RNA
TURNOVER
AND
PROTEIN
535
INTAKE TABLE
II
EFFECT OF PROTEIN INTAKE ON THE DAILY FRACTIONSL RATE OF CHANGE IN RAT LIVER RNA CONTENT, AND ITS RATES OF DEGRADATION
AND
SYNTHESIS 1 Daily
Expt’l series
IA
IB
II
Dieta
18% 0% 18% 0% 18yc 0% 0%
L
-!-r L Days
after
H3
- orot~c
ocld
FIG. 1. Total amount of RNA per liver and rate of loss of 3H-RNA from the livers of rats receiving a diet adequate in protein (O---O) and of rats adapted for several days to a protein-free diet (o-----O). The rats were fed equal amounts of either a diet containing 18% protein or a proteinfree diet. On the third day they were injected with erotic acid-3B. and measurements of total RNA and 3H-RNA per liver were made from 3 days after isotope administration onward. The amounts of total RNA and of 3H-RNA per liver are plotted on a logarithmic scale. Each point represents results from the pooled livers of three animals.
RNA per day, the fractional synthetic rate must have been less than that for degradation, namely, 14% per day. These estimates of rates of degradation and synthesis are set out in Table II, which also includes a replicate experiment in which only 10 g of each diet was fed per day. Essentially the same picture was obtained, namely, that the turnover of liver RNA slows down in protein deficiency, there being both retarded synthesis and retarded breakdown. The latter exceeds synthesis, so that a persistent slow loss of RNA occurs as a result of lack of protein in the diet. Second series of experiments. The objective here was to examine liver RNA turnover during the transition period from an ade-
Protein Protein (from days on) Protein Protein (from days on) Protein Protein (first days) Protein (from days on)
3
fractional
rate
(%)
Change in RNA
;y.g a rate
$1 - 3
-26 -17
+27
0 6
-23 -12
$23 +6
-21 -43
+22
-15
+10
3
-
2
+1 -10
3
-
5
(1 Fifteen grams of each diet were IA and II, and 10 g. in Expt. IB.
.-
Spthetic rate
+14
f33
fed in Expts.
quate protein intake to a protein-free diet, since previous studies (3, 4) showed that liver RNA content undergoes a considerable reduction at this time, and there was some evidence to suggest that this occurred because of accelerated RNA breakdown. Twenty-one rats of initial weight close to 230 g (range 230-237 g) were fed the 18% casein diet ad libitum for 3 days, and were then injected with 3H-orotic acid. The 18% casein diet was then fed for a further 2 days at a level of 15 g per day, after which three rats were killed to provide an initial level for the quantity of RNA in each liver and its isotope content at this point. The remaining rats were divided into two groups of equal average weight. One group continued to receive 15 g daily of the 18% casein diet, while the other group was started on 15 g daily of the protein-free diet. Groups of three rats from each diet were killed on the second, fourth, and seventh days after separating the animals into two groups. Figure 2 shows the changes produced in total liver RNA and in radioactivity retained in total ribosomal RNA, and the fractional rates of change computed from these data are summarized in Table II. The total RNA content of the livers of rats receiving the 18% pro-
536
ENWONWU
‘\
6 t
Days
afler
3H
-erotic
acid
FIG. 2. Total amount of RNA per liver and rate of loss of 3H-RNA from the livers of rats receiving a diet adequate in protein (@---a) and of rats immediately after changing to a protein-free diet (O-----O). All animals were fed the diet containing adequate amounts of protein until 2 days after erotic acid-3H administration; one group was then given a similar amount of a protein-free diet. The amounts of total RNA and of 3H-RNA per liver are plotted on a logarithmic scale. Each point represents results from the pooled livers of three animals.
tein diet rose slightly (+ 1% per day) during the experimental period. In agreement with the findings of previous investigators (3, 4), withdrawal of protein resulted in a loss of liver RNA that occurred in two phases. During the first 2 days, there was a sharp reduction at a fractional rate of 10% per day. Thereafter, the rate of loss slowed to some 5 % per day, similar to that found in the two experiments reported above, in both of which the rats were allowed to adjust for a few days to the low intake of protein before measurements of liver RNA content were made. The loss of radioactivity by the two groups of rats is also shown in Fig. 2. Rats on the 18% casein diet throughout the experiment showed a progressive decrease in total radioactivity in the liver RNA with a half-life of
AND
MUNRO
81 hr. Consequently, the fractional rate of degradation was 21% and the rate of synthesis was 22%, being thus similar to the turnover rates found on this diet in the other experiments shown in Table II. The rats that received the protein-free diet showed extensive changes in rate of turnover of RNA. During the first 2 days after deleting protein from the diet, degradation was considerably accelerated, and at all times thereafter the rats on the protein-free diet had less radioactive RNA in their livers than remained in the livers of animals receiving adequate amounts of dietary protein for a similar length of time (Fig. 2). During the first 2 days after withdrawal of dietary protein, the half-life of ribosomal RNA was computed to be 38 hr from the slope between Days 2 and 4 after 3H-orotic acid administration. Consequently, the fractional rate of RNA breakdown was 43 % per day, which is twice as fast as the rate of degradation on the 18% casein diet. Since the loss of RNA during this 2 day period was only 10% per day, the fractional rate of synthesis can be computed to be 33% per day. These estimates of turnover do not have a high degree of precision since they are based on only two points instead of the three or more points used for calculation of turnover for animals equilibrated in the diets. Nevertheless, it can be concluded that the immediate loss of liver RNA after deprivation of dietary protein is due to a sudden acceleration in rate of RNA breakdown. On continuing to feed the protein-free diet, degradation of RNA quickly slowed down to somewhat less than that observed for rats on the 18% casein diet, and the fractional rate of RNA synthesis also became even slower. These latter findings for rats conditioned to the protein-free diet agree with those observed in the earlier experiments recorded in Table II. Ribosome aggregation and protein intake. Since our studies showed a reduction in ribosomal RNA breakdown in the livers of animals receiving a protein-free diet for several days, it was of interest to determine the sizes of ribosome aggregates in the livers of these rats as compared with animals receiving an adequate intake of protein.
LIVER RNA TURNOVER 0.5 NORMAL .----
MALE
RATS
0% CASEIN 16 % CA~EIN
FOR 9 DAYS (IOg/RAT/DAY) FOR 9 DAYS (KI~/RAT/DAY)
i 0.4
t-
AND
PROTEIN
INTAKE
537
monosomes and disomes in the liver and the total concentration per g of liver is much lower in the case of the protein-deficient animals, due to extensive loss of RNA by this group. Evidence obtained from an earlier investigation (23, 24) shows that ribosome subunits are much fewer in the livers of rats that have received a proteinfree diet for several days. This infrequency of monosomes and subunits is of significance when we come to consider the mechanism regulating RNA breakdown. DISCUSSION
1 10% -40% TOP
SUCROSE
DENSITY
GRADIENT
1 BOTTOM
FIG. 3. Polysome profiles on sucrose gradients obtained from the livers of rats fed equal amount of either an 18% protein diet (----) or a proteinfree diet (--) for 9 days. The livers of four rats on each diet were combined and amounts of cytoplasmic extract equivalent to the same weight of liver from ea,ch group were put on each sucrose gradient.
of rats were fed 10 g of either the 18% protein diet or the protein-free diet for 11 days, and were killed on the morning of Day 12. The livers from individuals in each dietary group were pooled and analyzed. Those fed on the 18 % casein diet had 44 mg RNA per liver, whereas those on the proteinfree diet had 20 mg RNA/liver. Figure 3 shows the total polysome profiles obtained from the livers of animals on each diet. The smaller profile on the protein-free diet is due to the fact that polysomes prepared from equal weights of liver were layered onto each gradient, and the concentration of RNA per g of liver was less in the case of the rats on the protein-free diet (cf. Table I). The percentages of monosomes plus disomes in the two profiles were very little different in the two profiles (39% on the proteincontaining diet and 44 % on the protein-free diet); in consequence, the total number of Groups
The general characteristics of ribosomal RNA turnover in the liver have been examined by Hirsch and Hiatt (10). They examined the rate of loss of label from liver ribosomes after injection of both Wguanido-arginine to label ribosome protein and 3H-erotic acid to label RNA. The guanido group of free arginine is so quickly exchanged in the arginineeornithine cycle that the label is rapidly obliterated when guanido-labeled arginine is released from liver proteins. Consequently, this radioactive amino acid is used to obtain true halflives of liver proteins without significant recycling of the label (25). In Hirsch and Hiatt’s study, ribosomal protein and ribosomal RNA gave identical half-lives of 5 days; this indicates that the pyrimidine bases labeled from 3H-erotic acid are not significantly recycled, and, consequently, that this label provides a good approximation to the true rate of degradation of ribosomal RNA. Hirsch and Hiatt (10) also examined the effect of prolonged starvation on the rates of RNA degradation and synthesis, using changes in quantity of RNA per unit of DNA as well as loss of specific activity to make these calculations. They observed that the fractional rate of RNA degradation increased and the fractional rate of RNA synthesis decreased during starvation. This resulted in a rapid and extensive loss of RNA per cell, accompanied by a loss of lo-25 % of liver DNA, which would be indicative of some cell turnover. This large change in the amounts of DNA per liver due to starvation is contrary to the findings of others (1, 2) who reported no change in DNA over a
53s
ENWONWU
2-day period of starvation. In a recent paper (26), Hirsch et al. reported that their procedure for measuring liver DNA gives invalid results in starving animals. Consequently, the values for RNA turnover in starving animals calculated by Hirsch and Hiatt (10) may also require revision. Starvation involves withdrawal of all nutrients. In our present series of experiments, we have varied only intake of protein, since earlier studies (5, 6) had suggested that availability of amino acids affects rate of degradation of liver RNA. The present studies show that animals habituated to a diet deficient in protein undergo a reduction in rate of both synthesis and degradation of ribosomal RNA. The latter response is contrary to the effect of starvation observed by Hirsch and Hiatt (10). The diminished rate of RNA synthesis found in our proteindepleted animals contrasts with the increase in RNA polymerase activity observed in the liver nuclei of protein-depleted rats by Shaw and Fillios (27). No explanation can be offered at present for this discrepancy. The transition from a diet adequate in protein content to one deficient in protein is of special interest since there is an initial rapid loss of RNA from the liver at the time of change. The immediate effect of withdrawing protein from the diet is not a slowing down of degradation but a sudden acceleration for the first 2 days, followed by retarded degradation from then on. The immediate increase in degradation agrees with our earlier studies (4, 5) in which incorporation of 32Pand of 14C-glycine into the purines of liver RNA and its precursors was examined. During the first day of protein withdrawal, incorporation of 14C-glycine into RNA was greatly reduced, and this was shown to reflect similar labeling changes in the precursor pools of adenine and guanine compounds. The effect was attributed to filling of the precursor pools with breakdown products of RNA degradation (5) ; possibly this caused feedback inhibition of purine biosynthesis. As soon as this short, initial period of intense RNA degradation is over, the uptake of precursor into purines resumes again (5). Changes in acid-soluble purine labeling were also observed a few hours after
AND
MUNRO
a tryptophan-deficient mixture of amino acids was fed to rats in place of a complete amino acid mixture (as), and it was subsequently shown (6, 29) that the absence of tryptophan from the mixture causes an accumulation of monosomes and free ribosome subunits in the liver cell. Since we have shown that the free ribosome subunits are rich in endogenous ribonuclease (23), it was concluded (7) that tryptophan deficiency or general protein deficiency results in accumulation of subunits and thus in accelerated RNA breakdown, which proceeds until a new equilibrium between polysomes, monosomes, and subunits is established. This occurs after a few days of protein depletion, as shown by the absence of excess monosomes in the polysome profiles of protein-depleted animals (Fig. 3). On the basis of this hypothesis, the fractional rate of RNA degradation will be dependent on the subunit population of the cell. In fact, Munro et al. (24) found that the subunit fraction of the cell was most abundant during the period of rapid RNA loss during starvation, less for animals during absorption of a protein meal, and least after a few days of a protein-free diet. This change in subunit abundance thus correlates well with changes in the rate of loss of label from ribosomal RNA (Table II). It is of interest that the rate of RNA synthesis does not fall as soon as the protein-free diet is given, but only after extensive RNA loss has occurred (Table II). The nature of this control over synthetic rate is not known. REFERENCES 1. DAVIDSON, 2.
3. 4.
5. 6. 7. 8.
J. N., Cold Spring Harbor Symp. @ant. BioE. 12, 50 (1947). THOMSON, R. Y., HEAGY, R. C., HUTCHISON, w. c., AND DAVIDSON, J. N., Biochem. J. 63,460 (1953). KOSTERLITZ, H. W., J. Physiol. 106,194 (1947). MUNRO, H. N., NAISMITH, D. J., AND WIKRAMANAYAKE, T. W., Biochem. J. 64, 198 (1953). CLARK, C. M., NAISMITH, D. J., AND MUNRO, H. N., Biochim. Biophys. Acta 23,587 (1957). WUNNER, W. H., BELL, J., AND MUNRO, H. N., Biochem. J. 101, 417 (1966). MUNRO, H. N., Fed. Proc. 2’7, 1231 (1968). GERBER, G., GERBER, G., AND ALTMAN, K. I., J. Biol. Chem. a36, 2682 (1960).
LIVER
RNA
TURNOVER
9. LOEB, J. N., HOWELL, R. R., AND TOMKINS, G. M., Science 149, 1093 (1965). 10. HIRSCH, C. A., AND HIATT, H. H., J. Biol. Chem. 241,5936 (1966). 11. WILSON, S. H., AND HObGLAND, M. B., Bio&em. 1. 103, 556 (1967). 12. BLOBEL, G., AND POTTER, V. R., Biochim. Biophys. Acta 166, 48 (1968). 13. HADJIOLCIV, A. A., Biochim. Biophys. Acta 119, 547 (1966). 14. ROGERS, Q. R., AND HARPER, A. E., J. Nutr. 87, 267 (1965). 15. DRYSDALW, J. W., AND MUNRO, H. N., Biochim. Biophys. Acta 138, 616 (1967). 16. BRITTEN, R. J., AND ROBERTS, R. B., Science 131, 32 (1960). 17. BOCK, R. M., L4~~ LING, N. S., Anal. Chem. 26,1543 (1954). 18. READER, .R. W., AND STANNERS, C. P., J. Mol. Biol. 28, 211 (1967). 19. BRAY, G. A., Anal. Biochem. 1, 279 (1960).
AND 20.
PROTEIN MUNRO,
539
INTAKE H. N.,
AND
FLECK,
A., Analyst
91,78
(1966). 21. KECK, K., Arch. Riochem. Biophys. 63, 446 (1956). 22. HIRSCH, C. A., J. Biol. Chem. 242,2822 (1967). 23. HIRD, H. J., MCLEAN, E. J. T., BND MUNRO, H. N., Biochim. Biophys. Acta 87,219 (1964). 24. MUNRO, H. N., MCLEAN, E. J. T., AND HIRD, H. J., J. Nutr. 83, 186 (1964). 25. SWICK, R. W., J. Biol. Chem. 231, 751 (1958). 26. EDELMAN, M., HIRSCH, C. A., HIATT, H. H., AND Fox, M., Biochim. Biophys. Acta 179, 172 (1969). 27. SHAW, C., AND FILLIOS, L., J. Nutr. 96, 327 (1968). 28. MUNRO, H. N., AND CLARK, C. M., Biochim. Biophys. Acta 33, 551 (1959). 29. PRONCZUK, A. W., BALIGA, B. S., TRIANT, J., AND MUNRO, H. N., Biochim. Biophys. Acta 167, 204 (1968).