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The measurement of nucleic acid turnover in rat liver
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
63,
226-242
(1956)
The Measurement of Nucleic Acid Turnover in Rat Liver’, 2 Robert W. Swick, Arthur L. Koch3 and Dorothy T. Handa From the Argonne National Laboratory, Divisionof Biological and Medical Resarch, Lemont, Illinois; and from the Department of Biochemistry, University of Chicago, Chicago, Illinois Received
December
5, 1955
Although the metabolism of the nucleic acids has been extensively studied with many isotopic compounds and under a variety of experimental conditions, no satisfactory estimates of the rates at which these compounds are synthesized or degraded have been presented. The reasons for the absence of these data are manifold. First, when the rise in the radioactivity of the nucleic acids is observed after a single dose of tracer, it is necessary to identify and to measure the amounts of the intermediate compounds, and at various times their specific activities. Because these precursor pools are largely unknown, this method has not been useful for absolute measurements, although it has permitted comparisons of nucleic acid turnover under different physiological and pathological conditions. Secondly, when the fall in radioactivity of the nucleic acids is to be followed, the extent to which isotope, derived from the catabolism of either the nucleic acids or from other labeled body constituents, is reincorporated into the nucleic acids should be measured. This hitherto has not been possible. Thirdly, because in single-dose experiments the specific activity of the isolated compound is very much smaller than that of the administered compound, the danger of contamination of the former by traces of the lat,ter becomes important. Also, the possibility exists that the isotope is not 1 Work performed under the auspices of the U. S. Atomic Energy Commission. * A preliminary report was presented at the meetings of the American Society of Biological Chemists, San Francisco, Calif., April 11-15, 1955. 3 Present address: Department of Biochemistry, College of Medicine, University of Florida, Gainesville, Fla. 226
NUCLEIC
ACID
TURNOVER
227
uniformly distributed within the tissue; i.e., some cells in the tissue may have more of t’he tracer, or some nucleic acid components may become more highly labeled than others, either as the result of different turnover rates or of different activity in the precursor pools. In addition to these considerations it is, of course, necessary to separate completely the two types of nucleic acids from each other, to determine the isotope content of pure chemical compounds, and to design the experiments to reduce the effects of variation in the biological material. In the experiments to be described, C1402was used as a tracer and was usually administered continuously. When this isotopic compound is given, the intracellular specific activity rises immediately, remains at a constant value for the duration of the administration, and then drops abruptly t’o a negligible value (1). The continuous administration of the tracer compound eliminates transient changes in the specific activity of t’he intermediate pools, but the effects of feedback (i.e., reutilization of nucleic acid fragments) within the tissues remain. However, correcbion for feedback can be made with the help of the following consideration. It will be shown below that CO* contributes 1.12 moles of carbon for the synthesis of the purine ring: metabolic CO2 is utilized without dilution for the synthesis of the C-6 position (1.0 mole); it is diluted about eightfold for the synthesis of the carboxyl group of glycine (a), but the latter is used without further dilution for the C-4 position of the purines (0.12 moles). Hence from the specific activity of metabolic COZ at any time [which is equal to the specific activity of urea (l)], one may calculate the specific activity of purines synthesized de novo at that time, which quickly mix with those derived from the breakdown of nucleic acids, coenzymes, etc. Then from the difference between this calculated act’ivity and the observed specific activity of the RNA, the extent to which the immediate precursors of the nucleic acids are formed from the degradation of the RNA may be calculated. Such calculations are possible either for experiments in which highly radioactive, newly synthesized purines are diluted with unlabeled purine from nucleic acid breakdown or for experiment’s in which purines synthesized de novo are non-radioactive and the nucleic acid purines are labeled. We have thus been able to estimate the magnitude of a number of processes of nucleic acid turnover4 in both growing and nongrowing rats. 4 For the purposes of the present paper the definition of turnover must be extended to growing systems. Turnover has come to mean the concomitant and equal synthesis and degradation of a constituent in an organism in dynamic equilibrium.
228
SWICK,
KOCH
AND
HANDA
It is assumed that there are some small pools of adenine and guanine intermediates which are maintained by de novo synthesis and by the breakdown of ribonucleic acid (RNA). The contribution, if any, from the breakdown of deoxyribonucleic acid (DNA) must be small in comparison to RNA, because of the smaller amount of DNA present and because of its apparent stability (see below). It is further assumed that extrahepatic metabolism of nucleic acids is without effect on the isotope content of the liver. This would appear to be justified by the experiments of Dancis and Balis which show in several cases that activity present in the nucleic acids of one tissue is not transferred to other tissues (3). The following scheme, suggested by the literature and the data to be presented here, would appear to be a reasonable representation of nucleic acid metabolism, and one which lends itself to mathematical treatment. The symbol V represents the reaction velocity in pmoles of the given compound formed by the reaction per day; each capital letter, as indicated in the model, designates the specific activity of the compound at any time; and the lower-case letter represents the amount of this material present. Equations will be derived for the specific activities of the adenine in the two types of nucleic acid under several experimental conditions. Equations identical in form will be used for guanine. For the present However, it appears necessary
in this instance to define “turnover rate” as the fraction of the component that is either synthesized or degraded per unit time for all cases in which an experimentally determined “half-life” can be measured, whether growth occurs or not. This definition is operational and applicable whether the component in question is an isotopic atom; a pure chemical species; a mixture of chemical species; a fraction that is defined operationally, such as a subcellular fraction; or a whole cell, tissue, or “RNA,” “DNA,” or “protein”; organism. This definition of “turnover rate ” is equally applicable whether the component is produced or degraded by a completely random process or by a process in which formation of a new cell and its life history is involved. It also applies to a component consisting of parts which have different turnover rates although they are derived from a common precursor, but that may or may not be separable from each other. In this case the measurement must be conducted in a short enough time so that each part is weighted equally in the over-all measurement. In the case of the growing system, the growth rate of a component is equal to the difference between the rate of synthesis and the rate of degradation, the latter, then, being the measure of the turnover of the component. In cases where only synthesis occurs and degradation is negligible, there is no renewal and no turnover per se. However, there would still be defined a “turnover rate for synthesis” which would be equal to the growth rate of the component in question.
NUCLEIC
ACID
V 50 f I V ---=-+‘I
229
TURNOVER
guanine (G) vtc
excretion RKA guanine (R,)
F
DNA guanine (DC) V4C
co 2+ “purine” (PI
VA-0 V aa
T71A
,“adenine”(A)
R?U’A adenine (RA) DIVA adenine (DA)
v4.k B
V 5.4
excretion
purposes we will not consider the minor reaction pathway labeled Jr,-, but will introduce the pathway in considering the final results. Let us first consider the specific activity of the pool labeled “A” in the scheme, which corresponds to all of the various soluble adenine-containing compounds of the cell. Because the amount of these compounds is small compared to the amount of nucleic acids and because they are metabolically very active (4), their average specific activity must be a function only of t,he specific activities of t,heir precursors. In this case there are two quantitively important sources: de novo synthesis and t-he degradation of RNA; therefore the specific activity, A, will be the weighted average of P and R:
(subscripts referring to adenine, e.g. VIA, have been dropped for the purposes of this derivation). In the experiments wit’h growing rats the amounts of t,he nucleic acids are increasing continuously. In rat,s of the age used here t’he increase is very nearly linear. Therefore bhe amounts of Rn’A and DSA present at, any time are r = To + (V, -
V&
(2)
and d = do + VJt
(3)
230
SWICK,
KOCH
AND
HANDA
where the subscript 0 refers to the initial amount, and the velocities are considered to be constant during the course of the experiment. During the continuous administration of C!1402the specific activity of the purine precursor pool is maintained at the definite value P, equal to 1.12 times that of the metabolic COz as measured by the specific activity of the urea carbon. Then the total activity in the RNA is given by
d(rR) _ dt By differentiation tained :
Tr,A -
V,R = F
+ y
.
(4)
of Eq. (2), the change in the amount of RNA is ob-
and therefore, the change in the specific activity rdR -=V&-
VIR -
dt
of the RNA is given by
(V, - VJR = Vz(A - R).
(6)
Eq. (6) is also the equation for the adult nongrowing animal as can be seen by setting ‘cl’s= Vs in Eq. (4). Substituting the value of A from Eq. (1) into Eq. (6) we obtain: -rdR
dt
= ‘vz
VlV2
v3
Vl + v3
Vl
+
(7) v3
which simplifies to
If we let P represent the average amount of RNA present and consider it to be constant, we may then integrate Eq. (8) to obtain R = P[l
- exp(-
(9
v,~v,X~Xt)]
If, however, we do not use an average value for r but instead substitute from Eq. (2), Eq. (9a) is obtained. R =P[l
-exp(
-v1vz (VI + VJ(V, = P[l
- Vd In - (9
(
l+
‘+T))]
(V, + ii;::+
VJ
(gu)
NUCLEIC
ACID
231
TURNOVER
where the subscript f represents the final state. Eq. (9) closely approximates Eq. (9a). Even when r varies over a twofold range, the use of Eq. (9) rather than Eq. (9a) will result in an error of only 4 %. For further manipulation, however, the precise equation, (9a), is used. The rate of change of total acbivity of the DNA is given by &Dd dt Introducing D
= AV
4
-PV4T 4
+ lisR V
v1+
Eq. (9a), integrating, =
= ‘lp
773
and reintroducing [rm,
(Vlf
F:-
Vd x
(10)
4
Eq. (9u), we obtain
- P(rf - ro)] 6
(11)
Thus the specific activity of the DNA is given by the difference of two terms. The first is the specific activity in the absence of RNA turnover and the second is a correction for t’he formation of less active DNA precursors from the RNA. In another experiment, the continuous administration of isotope was stopped, and the changes in activity of the nucleic acids were noted after an interval of time. We may assume that P = 0, since after cessation of administration the activity of the met’abolir CO? drops abruptly to a negligible value. In this case, Eq. (6) becomes rdR __ dt which on integration
= vz(A _ R) = f$
R 1
(12)
3
yields It = Roexp -
VII-,t (Vl + Vig)f=
or more exactly
1
R = R”[ l - 0: (V,+ i3;;: + V,) where R. is the initial Eq. (10) becomes
specific activity
in the RNA. Then for the DlSA,
d DO = A-VI = v, v3v4+ v3 x R. dt SubstJitutjing Eq. (13a) into Eq. (14) and integrating, UWf
(13a)
(14) we obtain
= @do + vl + ;; _ Tit (roRo - r&J
(15)
232
SWICK,
KOCH
AND
HANDA
which can be rearranged to give v4 A(Dd) -- A@6 = v, + vs - Vz’
(16)
Thus we have obtained equations predicting the isotope content of the nucleic acids at any time for these two different types of experiments in terms of reaction velocities and amounts of the component present. The values for these reaction velocities will be calculated from the experimental data to be presented. Usually, however, the results of metabolic studies are expressed either in terms of “half-lives” or turnover rates,4 the two being related by the fact that their product is 0.693 or In 2. Actually, the calculation of turnover rates has the advantage over the calculation of reaction velocities in that fewer experimental quantities are necessary. In the case of the nongrowing animal in “dynamic equilibrium,” only the data on the specific activity of a component is required for calculation of turnover rates. Even in the growing animal certain quantities having the dimensions of a turnover rate may be computed from the specific activity data and the extent of growth. For the present model three kinds of turnover rates may be obtained. The first is V,V,/(V, + V,)P, which gives the apparent rate of turnover of RNA. This quantity may be calculated from Eqs. (9) and (13). The other two quantities require a knowledge of the degree of growth. In the experiments reported here, the initial weight of the liver of the animal is estimated from the weights of the livers of animals of comparable age, and thus the degree of increase in liver weight is accurately known. The ratio of the final to initial liver weight (L) is given in Table I. If we assume that the increase in various components is proportional to L, then Eq. (16) becomes LDI - Do = v4 VI + vs Ro - LR
v, x a
(17)
and Eq. (11) becomes DL I.+1
_= d v4
v4
+ VI + vo -
vz PT
1 (18)
Thus from specific activity data and the factor of growth, the quantity [V4IWl + v3 - Vdl x (eo is obtained, and from this value Eq. (18)
XUCLEIC
233
ACID TURNOVER
yields VJd which is the rate of DNA synthesis. The quotient of Vdd divided by [ VJ( VI + VB - V,)] X (f/d) is (VI + VS - VJ/? or the third turnover rate. It should be noted, however, that in the case of “dynamic equilibrium” this becomes VI/r. Actually, [VJ(V, + VS- VZ)] X (f/d) seems to be the more interesting quant,ity because it, is related to the conversion of RNA label to DNA. The primary goal of the computation is to calculate these turnover rates. Then, secondarily, from estimates of the amounts of various components, individual reaction velocit,ies may be calculated for the various processes indicated in the scheme. If the growth rate was constant during the experiment,al period, the reaction velocities must have been constant also; but if deviation in growth rate has occurred, the calculated reaction velocGt,ies will be t’he average values. METHODS Animals were fed, at hourly intervals, purified diets containing CaC1”03 (Table I) (2) for periods ranging from 7 to 59 days, thus maintaining the radioactivity of the metabolic CO? at a constant level as established by the determination of t,he specific activity of the excret,ed urea. The nucleic acids were isolated by t,he proIf care is taken cedure described by Tyner et al. (5) with only slight modifications. to have all solutions at 0” before the addition of acid, the RNA fraction should be devoid of cont,amination with DNA. (A negative Dische test indicated less than 2% contamination.) In order to obtain DNA free of RNA, the DNA fraction was hydrolyzed to free bases and apurinic acid (6) by heating at 100” at pH 3 for 1 hr. Under these conditions the labile bond between the purines and deoxyribose phosphat,e is broken, while that between purines and any ribose phosphate present is only slightly affected as shown by the failure of these conditions to hydrolyze significant amounts of adenylic acid in control experiments. When the hydrolyzate is passed through a Dowex 2 Cl- column at a slightly acid pH, the nucleotides are retained, permitting the isolation of the free DNA purines. After separation of all the purines by cation exchange on Dowex 50 H+ (7), extraneous carbon compounds were eliminated by isolating adenine and guanine as the silver salts and then reconverting t,o the free purines. When adenine was degraded by heating with H&J1 to 190” for 1 hr. in a closed, C&free system under 60 cm. of NZ , I he as C6 position was liberated as CO* , the C-4 and C-5 cnrl)ons were obtained glycine, and C-2 and C-8 were converted to CO.5 The CO, was collected in liquid X;2 , and the glycine was isolated and decarboxylated wit’h ninhydrin to yield COZ Adenine and guanine were converted to CO2 by wgt combustion, and the specific radioactivity of all preparations was measured by proportional gas counting (8) with a standard deviation of ~1%. The weight of the liver at the beginning of the desired experimental period 5 Personal University.
communication
from
Dr.
G. Robert
Greenberg,
Western
Reserve
234
SWICK, KOCH AND HANDA
which was requisite for the estimation of growth, was obtained by sacrificing a number of rats of similar body weight and calculating a regression value for the rats used in the experiment. The standard error of the predicted value was calculated according to Tippett (9).
RESULTS
The results of the isotope analyses are present in Table I. Experiment A indicates that after an adult rat has been exposed to isotopic COz for 59 days the RNA guanine (and presumably adenine as well) has reached the theoretical maximum specific activity: 112 % of the urea value. On degradation of RNA adenine (Expt. C), more than 98 % of the radioactivity was accounted for in the C-6 and C-4 positions, as predicted, Of t.he isotope, 11% was in t,he C-4 position, which is derived from the carboxyl group of glycine, while 89 % of the total activity was in the C-6 position. Therefore, as the same distribution of activity must occur in the adult rat, the C-6 position has a specific activity of 89 % X 112 % = 100 % of the urea value. Therefore it may be concluded that COz is the sole precursor of the C-6 posit’ion; i.e., there is no dilution of the metabolic COa used for the synthesis of this position. As mentioned above, the specific activity of the 4-position is about 12 % of the metabolic CO2 , and this value is identical to that obtained for the carboxyl group of glycine from liver protein in this same animal (2). It can therefore be concluded that glycine is the sole precursor of the 4,5,7-positions of the purine because both the RNA and liver protein were at isotopic saturation in this animal.6 In Expt. A the specific activity of the DNA purines was slightly less than one-half of the specific activity of the RNA purines, indicating that 59 days is less than the apparent half-life of DNA. Experiments B, C, and D show that in growing rats the RNA purines are not isotopically saturated after 7-9 days exposure, and that considerable activity is associated with the DNA compounds. Since the liver weights have approximately doubled during the exposure period, one would expect the DNA to be approximately half-saturated if new growth and DNA synthesis are concomitant. This seems to be the case. Experiments D and E wereperformed to evaluate the conversion of adenine into guanine and their transfer from RNA to DNA. During the sec6 One may also conclude that RNA, like protein of rat liver (2)) consists entirely or almost entirely of substances that are being actively metabolized. It is of interest that the turnover rates of the two components are similar, although protein is present in much larger amounts.
Treatment
in liver
was calculated
in live
I
398 353
848 210
1068 91
118 53
1426 1439
-
1.87 2.22*
-
for the second period.
1.44a
I.610
8.9 1.3
-
1.2
10.3
71
94
65
122
236
129
316
318
394
2.30
181
176
-
403
326
2.20
1.07
84.5
RNA
in 100 counts/min./mmole
189
activity
212
168
Specific
in Liver
4denine
Acids
Adenine
6) Final wt. Initial
wrease
TABLE I and the Radioactivity of Nucleic
Constant exposure: 59 days; 2.2 rc./g. of amino acid diet (2) Constant exposure: 9 days; 5 pt./g. of 18% casein diet Constant exposure: 9 days; 5 pt./g. of 18% casein diet Constant exposure: 7 days; 16.8 pt./g. of 18% casein diet Constant exposure: 7 days; 16.8 pt./g. of 18% casein diet. No activity: 19 days; 12% casein diet Short exposure : 1.16 mc.; 120/, casein diet 21 days Short exposure: 1.16 mc.; 12% casein diet 21 days Short exposure: 1.16 mc.; 12% casein diet 21 days; 18% casein diet 21 days Short exposure: 1.16 mc.; 12% casein diet 21 days; 18% casein diet 21 days
0 Per cent increase
I. Weanling
H. Weanling
G. Weanling
P. Weanling
E. Weanling
D. Weanling
C. Weanling
B. Weanling
A. Adult
Animal
Growth
DNA
cpd.
65.4
43.8
103
238
333
278
128
-
81
236
SWICK,
KOCH
AND
HANDA
ond period (19 days), the specific activity of the RNA purines decreased sharply, in keeping with the known rapid turnover of RNA. That the specific activity of adenine decreased more rapidly than the specific activity of guanine is to be expected from the known ability of the rat to convert adenine into guanine but not the reverse; hence it appears that the radioactivity of RNA adenine is liberated by turnover and reincorporated into RNA guanine. The specific activity of the DNA purines did not fall to half of the original value during the second phase, as would have been predicted from the growth of the liver, indicating that RNA compounds contribute to the precursor pools from which DNA is synthesized. Each of the four rats of Expts. F-I was given 1.16 millicuries (me.) of NaHC1403 in three portions by intraperitoneal injection during the course of 1 day; two were sacrificed 21 days later; the other two, 42 days after the administration of radioactivity. In contrast to the reproducibility of the data obtained in the earlier experiments, there was a marked disagreement between similarly treated animals in the single-dose experiments. That the animals received identical amounts of Cl4 is evident from the similarity of the specific activities of the liver and muscle protein fractions of these animals (Table II). These discrepancies might result from variations between animals in the rates of renewal of RNA, or from variation in the rate of synthesis of nucleic acids, which are averaged out in constant exposure experiments but not in acute exposure studies. Our intention in the latter experiment was to measure changes in the radioactivity of DNA at a time when the contribution of radioactivity by RNA turnover would be minimal. During the first 21-day period the radioactivity of the RNA had decreased sufficiently to make only a small contribution to the DNA synthesized during the second phase, thus permitting a possible distinction between the dilution of DNA radioactivity by growth and any loss of DNA activity, for example, by turnover or cell death. In spite of the large variation between duplicate animals, there
Specific
TABLE II Activity of Tissue Proteins
Time si;Fyexposure, Experiment
F G H I
21 21 42 42
MUSCk,
Counts/min/mmole
4818 4916 1936 1570
C
Liver, counts/min/mmole
1177 1014 189 164
C
NUCLEIC
ACID
TABLE Apparent Experiment
l’wnover
III
Rate Constants
Equation No.
237
TURNOVER
of RNA
Adenine per da)
VI --.- X2 _____ VI + 1’0 r I Guanine per day
B
9
0.143
-
c I>
9 9
0.143 0.160 --
0.144 0.108
0.149
0.126
Av. D+E
13
0.129
0.074
F+H F+I G+H G+I
13 13 13 13
0.123 0.228 0.085 0.187
0.138 0.240 0.092 0.193
0.156
0.166
Av.
is some decrease in DNA specific activity solely by growth.
which cannot be accounted for
DISCCSSIOK
In Table III are shown the results of the esbimation of the apparent, rate of RNA turnover: VIVz/(Vl + I’,)?. This is only the net rate at which isotope enters or leaves RNA, and it includes the processes wherein RNA is broken down to yield fragments which are reutilized for RXA synthesis. The first group of estimates was obtained from experiments in which animals were exposed continuously to t,he isotope. The other estimates mere obtained from interanimal comparisons of t,he decrease in activit’y after administration of isotope had ceased, and, therefore, are less accurat,e. From these data it is apparent that the bulk of the Rn’A of liver is renewed at the same rate. At t,he end of the first 21-day period in Expts. F-I, the act’ivity of the RNA had decreased 25-fold. If even a small fraction of the RNA had a markedly smaller turnover rate (longer half-life) than the remainder of the RNA, the act,ivit,y in this hypothetical fraction would have decreased much less and the turnover measured during the next 21 days would he mainly of this component. Since the t\vo average estimates were the same, we may conclude that there is no such fraction. The presence of a small component, which is renewed more rapidly than the major port,ion of the RNA is, of course, not excluded.
238
SWICK,
KOCH
AND
HANDA
TABLE IV Puke
Composition
of Rat
Liver moles/g. liver
RNA DNA
Adenine Guanine Adenine Guanine
3.06 4.97 0.85 0.57
The other conclusion that may be drawn from these data is that intermediate adenine is a more efficient precursor of intermediate guanine than is guanine of adenine, in accordance with the observations of Brown et al. (10). This follows from the fact that the calculated turnover rates for adenine are higher than those for guanine in the continuous exposure experiments and lower in the decay experiments. This is to be expected if the reactions RNA adenine -+ intermediate adenine + intermediate guanine + RNA guanine are faster than the reverse reactions. We shall defer consideration of the significance of the quantitative estimate of VIVz/(Vl + V,)f to a later portion of the paper. The quantity V,/(V, + Vs - V,) is essentially the probability of feedback of an RNA purine molecule into the DNA. This quantity may be computed using Eq. (17), the data of Expts. D and E, and the ratio of purines present in these animals in the two types of nucleic acid. In Table IV are presented the amounts of the various purines found; each value represents the average of those determinations for which we were reasonably certain that the substances were obtained not only in radiochemically pure form, but also quantitatively. They are in substantial agreement with other values in the literature. There is considerably more RNA than DNA, and guanine predominates in the RNA, while adenine predominates in DNA. The absolute values fluctuate considerably, but the ratio between components was much less variable. Assuming that the ratio of RNA to DNA adenine is 3.6 and of guanine is 8.75, we obtain values for V,/(V, + V, - V,) of 12.1 and 13.45%, respectively. With these values one may then utilize Eq. (18) to calculate for growing rats the quantity VJd. This value should be relatively independent of inaccuracies in the estimates of material and also independent of the size of the animals; it is in fact the growth rate of liver DNA. Table V lists values of V,/d based on radioactivity measurements as well as those based on growth rate of liver (estimated from weight data). The estimates from the tracer data never exceed those obtained from liver weights; this fact leads to the following conclusions. First, the rate of
NUCLEIC
ACID
TABLE Estimate Expt. H
c D
V
of Growth
Rate [V.Jdl
From tracer data Adenine per day Guanine per day
0.07 0.056 0.062
239
TURNOVER
0.072 0.045
From liver weight per day
0.084 f 0.087 f 0.087 f
.004 .003 .012
cell death and DNA degradat’ion is insignificant compared with synbhesis in these growing rats. The est,imates of v.Ja for guanine are lower than for adenine, presumably for the same reason as they were for IISA. Scoondly, the hypothesis of Stevens et al. (11) and others (12), t,hat, when a cell divides both daughter cells receive only new DKA, is incorrect. This hypothesis requires that, under the conditions of our experiment in which the liver weights doubled (i.e., on the average, every cell divided once), there be a complete replacement of the DnTA; hence the rat,e of formation of DKA would have to be double the rate of growth of the liver. Since the rate of formation of DNA was similar to the rate of growt’h, we conclude, along with others (13, 14) that mit,osia is accompanied by the synthesis of only one cell complement of DNA. The growth rates estimated from tra.cer data are somewhat smaller thaii the mass growth rate. This discrepancy may be due to inadequacies of the model, although other models tested also yielded the same correction terms. On the other hand, it may be that the amount of DNA per unit weight is decreasing during the course of these experiments. Fukuda and Sibatani (15) have found that under the conditions of their experiments the DNA concentration is relatively caonstant in the age range used hew. However, in slightly younger animals they found a decrease in DXA concent’rat’ion of a magnitude which would account for the discrepancy in the present data. From 17Jd and VJ(V, + I’, - 1’2) X T,ld we may calculate the third turnover rate (Ii1 + I73 - V,)/?. From these three different turnover rates, t’he analytical data of Table IV, and the average rate of growt’h observed in these experiments, it is possible to calculate the individual reaction velocities V, through Ti6 for the nucleic acids of the growing rats. Average results for the young rats used in this study are tabulated in Table VI. Although in some instances the values are only approximate, hhey serve to indicat,e the relative magnitude of the processes involved. We have found that 4.9 kmoles of purine is synthesized and ‘L.-l pmoles is degraded in the liver of a 70-g. growing rat each day; 5.4 pmoles of purine
240
SWICK,
KOCH
AND
HANDA
.
TABLE Parameters
of Nucleic
VI
Acid Synthesis
Adenine, pm&s/day
2.6 1.8 1.0 0.22 1.6
VI
VP V3 V4 VS
in Young Rats” Guanine, pmoles/day
2.3 3.6 2.3 0.14 0.85
D It is assumed that the composition of the rat liver nucleic acid is that given in Table IV and that the average weight of the rat is about 70 g. (liver weight 3 g.).
is incorporated and 3.3 pmoles of purine is released from the RNA each day; 0.36 pmole of purine is incorporated into the DNA. Thus the rate of synthesis of new RNA exceeds the rate of de novo synthesis of purines. The expression of these results in terms of the concept of “half-life” would be inappropriate for two reasons. First, the apparent turnover rate of RNA, VIVJ(Vl + V,)P (Table III) is not only the rate of renewal of the nucleic acids but also includes the breakdown and resynthesis from fragments in the intermediary pools. The value of this quantity, in this case, corresponds to a half-life of about 4.6 days. This is comparable to previous estimates by Furst et al. (16), Bennett (17), and Bendich (18) for the adult rat. Secondly, two “half-lives” may be defined for every component in a growing animal. If one defines the half-life as that time required to synthesize at the observed rate an amount of RNA equal t’o 69.3% of that already present in the tissue, then the half-life is measured by 0.693r/Vz and is a considerably smaller quantity: 3.5 days as calculated from the present data. On the other hand, the time required to degrade at the observed rate an amount of the component equal to 69.3 % of that present in the tissue is of necessity longer in the growing animal. From the data presented here, this quantity is 6.4 days. Also, in the adult, nongrowing rat the use of the half-life concept for DNA turnover would be misleading. We have shown that DNA synthesis and cell growth
are concomitant
in the young
growing
rat, hence
it seems reasonable that DNA degradation is an aspect of cell death. We may interpret the DNA turnover as “cellular turnover,” with the probability of cell death either independent of its length of life, or dependent, with kinetics like those associated with the mortality of animal populations, normal red blood cells (19), or leucocytes (20). Although it is likely that both processes are involved,
the estimates
of the rate of replacement
of
DNA obtained in the two cases would differ by only 20 %. Selecting the second of the two alternatives, which assumes that no labeled DNA is de-
MJCLEIC
ACID
TURKOVER
241
graded during the experimental period, we may t’hen use Eq. (11) to calculate that the adult, nongrowing animal of Expt. A replaces 0.82 % of its DNA adenine and 0.82 % of its DNA guanine per day. In other words, slightly less than 1% of the DNA of cells of the rat liver is replaced daily: this estimat,e is of the same order as that calculated from the measurements of mit’otic activity by various workers (0.15-l % per day) (21,22), which suggests that intracellular turnover of DXA in living cells is either a much smaller or a non-existent process. It is well known that even the adult rat continues to grow very slowly; in this experiment, the extent of its growt,h (7 % in 59 days) account,s for 15 % of the DKA synthesized per day, leaving about 65 % of the DK:< formed per day for the replacement of dead cells. If all liver cells have the same life expectancy it could be inferred that the average life would l)e the reciprocal of this latter figure, or 150 days.’ These experiments are in complete accord with those recently presented by Fresco et al. (23). These authors administered adenine-XI5 and glycine-2-W to partially hepat,ectomixed rats and observed no appreciable loss of isotope from DSA between the first and fourt’h months after administrat,ion. Because regenerating rat liver was used, large amounts of radioactivit,y were incorporat’ed into all calassesof cells, but by the end of t,he first month the act,ivity in the RSA in all cells and in the DKA of the short-lived cells had been reduced. Since the cells that, contained radioactive DNA had all been produced veryshortlyaft’erhepatwt,omy, it may be concluded from their data, and in accord with our findings, that) the average life span of hepa,tic ~11s is great’er t’han 4 months.
The incorporation of CY402into the nucleic acids of rat liver has been st’udied by using the technique of cont’inuous administration of isotope. i The 150-day life span clearly represents an average for all liver cells. Presumably the life span of the hepatic cell is somewhat longer than 150 days and those of some of the other cell types may be considerably shorter. As mentioned above, 1here is significant loss of radioactivity from the DNA between the 3rd and the 6t.h week following the short exposure to C”O, . After correction is made for the small contribution of RNA to DNA in Expts. P-I, it may be calculated that very roughly 50% of the DNA activity present at the end of the 3rd week had disappeared by the end of the 6th week. Since the incorporation of isotope in the various types of cells should be inversely proportional to the life span of the class, one would infer that approximately 10% of the cells in the liver had a life span bet,ween 3 and 6 weeks.
242
SWICK,
KOCH
AND
HAND.4
The following conclusions were drawn: 1. Metabolic CO2 enters the C-6 position of the purines without dilution. Glycine enters the C-4, C-5, and N-7 positions, also without dilution. 2. In the young growing rat the percentage of new liver DNA synthesized per day is similar to the percentage increase in liver weight. Thus the synthesis of only one cell complement of DNA is associated with each mitosis, and the existing DNA is retained. 3. In young, rapidly growing rats, the rat,e of RNA renewal is rapid and all of the RNA is renewed at the same rate. 4. In the adult rat liver, the average life span of the DNA molecule is estimated to be at least 150 days. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23
BUCHANAN, D. L., J. Gen. Physiol. 34, 737 (1951). SWICK, R. W., AND HANDA, D. T., J. Biol. Chem. 218,577 (1956). DANCIS, J., AND BALIS, M. E., J. Biol. Chem. 207,367 (1954). BENNETT, E. L., AND KRUECKEL, B. J., Biochim. et Biophys. Acta 17,503 (1955); 17, 515 (1955). TYNER, E. P., HEIDELBERGER. C., AND LEPAGE, G. A., Cancer Research 13, 186 (1953). TAMM, C., HODES, M. E., AND CHARGAFF, E., J. Biol. Chem. 196, 49 (1952). HURLBERT, R. B., AND POTTER, V. R., J. Biol. Chem. 196,257 (1952). BUCHANAN, D. L., AND NAKAO, A., J. Am. Chem. Sot. 74, 2389 (1952). TIPPETT, L. H. C., “The Methods of Statistics,” p. 265. John Wiley and Sons, New York, 1952. BROWN, G. B., ROLL, P. M., PLENTL, A. A., AND CAVALIERI, L. F., J. Biol. Chem. 1’73, 469 (1948). STEVENS, C. E., DAOUST, R., ASD LEBLOND, C. P., J. Biol. Chem. 20% 177 (1953). Indicators,” p. 325. Interscience Publishers, Inc., HEVESY, G., “Radioactive New York, 1948. BARTON, A. D., Federation Proc. 13, 422 (1954). KIHARA, H. K., AND SIBATANI, A., Biochim. et Biophys. Actn 17, 579 (1955). FIJHUDA, M., AND SIBATANI, A., J. Biochem. (Japan) 40, 95 (1953). FURST, S. S., ROLL, P. M., AND BROWN, G. B., J. Biol. Chem. 183, 251 (1950) BENNETT, F,. L., Biochim. et Biophys. Acta 11, 487 (1953). BENDICH, A., Exptl. Cell Research, Suppl. 2, 181 (1952). LONDON, I. M., SIIEMIN, D., WEST, R., AND RITTENBERC, D., .I. Biol. Chem. 179, 463 (1949). KLINE, D. L., AND CLIFFTON, E. E., Science 116,9 (1952). BRUES, A. M., AND MARBLE, B. B., J. Exptl. Med. 66, 16 (1937). WIDNER, W. R., STORER, J. B., AND LCSHBAUQH, C. C., Cancer Research 11, 877 (1961). FRESCO, J. R., BENDICH, A., AND RUSSELL, P. J., JR., Federation Proc. 14,214 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
63,
226-242
(1956)
The Measurement of Nucleic Acid Turnover in Rat Liver’, 2 Robert W. Swick, Arthur L. Koch3 and Dorothy T. Handa From the Argonne National Laboratory, Divisionof Biological and Medical Resarch, Lemont, Illinois; and from the Department of Biochemistry, University of Chicago, Chicago, Illinois Received
December
5, 1955
Although the metabolism of the nucleic acids has been extensively studied with many isotopic compounds and under a variety of experimental conditions, no satisfactory estimates of the rates at which these compounds are synthesized or degraded have been presented. The reasons for the absence of these data are manifold. First, when the rise in the radioactivity of the nucleic acids is observed after a single dose of tracer, it is necessary to identify and to measure the amounts of the intermediate compounds, and at various times their specific activities. Because these precursor pools are largely unknown, this method has not been useful for absolute measurements, although it has permitted comparisons of nucleic acid turnover under different physiological and pathological conditions. Secondly, when the fall in radioactivity of the nucleic acids is to be followed, the extent to which isotope, derived from the catabolism of either the nucleic acids or from other labeled body constituents, is reincorporated into the nucleic acids should be measured. This hitherto has not been possible. Thirdly, because in single-dose experiments the specific activity of the isolated compound is very much smaller than that of the administered compound, the danger of contamination of the former by traces of the lat,ter becomes important. Also, the possibility exists that the isotope is not 1 Work performed under the auspices of the U. S. Atomic Energy Commission. * A preliminary report was presented at the meetings of the American Society of Biological Chemists, San Francisco, Calif., April 11-15, 1955. 3 Present address: Department of Biochemistry, College of Medicine, University of Florida, Gainesville, Fla. 226
NUCLEIC
ACID
TURNOVER
227
uniformly distributed within the tissue; i.e., some cells in the tissue may have more of t’he tracer, or some nucleic acid components may become more highly labeled than others, either as the result of different turnover rates or of different activity in the precursor pools. In addition to these considerations it is, of course, necessary to separate completely the two types of nucleic acids from each other, to determine the isotope content of pure chemical compounds, and to design the experiments to reduce the effects of variation in the biological material. In the experiments to be described, C1402was used as a tracer and was usually administered continuously. When this isotopic compound is given, the intracellular specific activity rises immediately, remains at a constant value for the duration of the administration, and then drops abruptly t’o a negligible value (1). The continuous administration of the tracer compound eliminates transient changes in the specific activity of t’he intermediate pools, but the effects of feedback (i.e., reutilization of nucleic acid fragments) within the tissues remain. However, correcbion for feedback can be made with the help of the following consideration. It will be shown below that CO* contributes 1.12 moles of carbon for the synthesis of the purine ring: metabolic CO2 is utilized without dilution for the synthesis of the C-6 position (1.0 mole); it is diluted about eightfold for the synthesis of the carboxyl group of glycine (a), but the latter is used without further dilution for the C-4 position of the purines (0.12 moles). Hence from the specific activity of metabolic COZ at any time [which is equal to the specific activity of urea (l)], one may calculate the specific activity of purines synthesized de novo at that time, which quickly mix with those derived from the breakdown of nucleic acids, coenzymes, etc. Then from the difference between this calculated act’ivity and the observed specific activity of the RNA, the extent to which the immediate precursors of the nucleic acids are formed from the degradation of the RNA may be calculated. Such calculations are possible either for experiments in which highly radioactive, newly synthesized purines are diluted with unlabeled purine from nucleic acid breakdown or for experiment’s in which purines synthesized de novo are non-radioactive and the nucleic acid purines are labeled. We have thus been able to estimate the magnitude of a number of processes of nucleic acid turnover4 in both growing and nongrowing rats. 4 For the purposes of the present paper the definition of turnover must be extended to growing systems. Turnover has come to mean the concomitant and equal synthesis and degradation of a constituent in an organism in dynamic equilibrium.
228
SWICK,
KOCH
AND
HANDA
It is assumed that there are some small pools of adenine and guanine intermediates which are maintained by de novo synthesis and by the breakdown of ribonucleic acid (RNA). The contribution, if any, from the breakdown of deoxyribonucleic acid (DNA) must be small in comparison to RNA, because of the smaller amount of DNA present and because of its apparent stability (see below). It is further assumed that extrahepatic metabolism of nucleic acids is without effect on the isotope content of the liver. This would appear to be justified by the experiments of Dancis and Balis which show in several cases that activity present in the nucleic acids of one tissue is not transferred to other tissues (3). The following scheme, suggested by the literature and the data to be presented here, would appear to be a reasonable representation of nucleic acid metabolism, and one which lends itself to mathematical treatment. The symbol V represents the reaction velocity in pmoles of the given compound formed by the reaction per day; each capital letter, as indicated in the model, designates the specific activity of the compound at any time; and the lower-case letter represents the amount of this material present. Equations will be derived for the specific activities of the adenine in the two types of nucleic acid under several experimental conditions. Equations identical in form will be used for guanine. For the present However, it appears necessary
in this instance to define “turnover rate” as the fraction of the component that is either synthesized or degraded per unit time for all cases in which an experimentally determined “half-life” can be measured, whether growth occurs or not. This definition is operational and applicable whether the component in question is an isotopic atom; a pure chemical species; a mixture of chemical species; a fraction that is defined operationally, such as a subcellular fraction; or a whole cell, tissue, or “RNA,” “DNA,” or “protein”; organism. This definition of “turnover rate ” is equally applicable whether the component is produced or degraded by a completely random process or by a process in which formation of a new cell and its life history is involved. It also applies to a component consisting of parts which have different turnover rates although they are derived from a common precursor, but that may or may not be separable from each other. In this case the measurement must be conducted in a short enough time so that each part is weighted equally in the over-all measurement. In the case of the growing system, the growth rate of a component is equal to the difference between the rate of synthesis and the rate of degradation, the latter, then, being the measure of the turnover of the component. In cases where only synthesis occurs and degradation is negligible, there is no renewal and no turnover per se. However, there would still be defined a “turnover rate for synthesis” which would be equal to the growth rate of the component in question.
NUCLEIC
ACID
V 50 f I V ---=-+‘I
229
TURNOVER
guanine (G) vtc
excretion RKA guanine (R,)
F
DNA guanine (DC) V4C
co 2+ “purine” (PI
VA-0 V aa
T71A
,“adenine”(A)
R?U’A adenine (RA) DIVA adenine (DA)
v4.k B
V 5.4
excretion
purposes we will not consider the minor reaction pathway labeled Jr,-, but will introduce the pathway in considering the final results. Let us first consider the specific activity of the pool labeled “A” in the scheme, which corresponds to all of the various soluble adenine-containing compounds of the cell. Because the amount of these compounds is small compared to the amount of nucleic acids and because they are metabolically very active (4), their average specific activity must be a function only of t,he specific activities of t,heir precursors. In this case there are two quantitively important sources: de novo synthesis and t-he degradation of RNA; therefore the specific activity, A, will be the weighted average of P and R:
(subscripts referring to adenine, e.g. VIA, have been dropped for the purposes of this derivation). In the experiments wit’h growing rats the amounts of t,he nucleic acids are increasing continuously. In rat,s of the age used here t’he increase is very nearly linear. Therefore bhe amounts of Rn’A and DSA present at, any time are r = To + (V, -
V&
(2)
and d = do + VJt
(3)
230
SWICK,
KOCH
AND
HANDA
where the subscript 0 refers to the initial amount, and the velocities are considered to be constant during the course of the experiment. During the continuous administration of C!1402the specific activity of the purine precursor pool is maintained at the definite value P, equal to 1.12 times that of the metabolic COz as measured by the specific activity of the urea carbon. Then the total activity in the RNA is given by
d(rR) _ dt By differentiation tained :
Tr,A -
V,R = F
+ y
.
(4)
of Eq. (2), the change in the amount of RNA is ob-
and therefore, the change in the specific activity rdR -=V&-
VIR -
dt
of the RNA is given by
(V, - VJR = Vz(A - R).
(6)
Eq. (6) is also the equation for the adult nongrowing animal as can be seen by setting ‘cl’s= Vs in Eq. (4). Substituting the value of A from Eq. (1) into Eq. (6) we obtain: -rdR
dt
= ‘vz
VlV2
v3
Vl + v3
Vl
+
(7) v3
which simplifies to
If we let P represent the average amount of RNA present and consider it to be constant, we may then integrate Eq. (8) to obtain R = P[l
- exp(-
(9
v,~v,X~Xt)]
If, however, we do not use an average value for r but instead substitute from Eq. (2), Eq. (9a) is obtained. R =P[l
-exp(
-v1vz (VI + VJ(V, = P[l
- Vd In - (9
(
l+
‘+T))]
(V, + ii;::+
VJ
(gu)
NUCLEIC
ACID
231
TURNOVER
where the subscript f represents the final state. Eq. (9) closely approximates Eq. (9a). Even when r varies over a twofold range, the use of Eq. (9) rather than Eq. (9a) will result in an error of only 4 %. For further manipulation, however, the precise equation, (9a), is used. The rate of change of total acbivity of the DNA is given by &Dd dt Introducing D
= AV
4
-PV4T 4
+ lisR V
v1+
Eq. (9a), integrating, =
= ‘lp
773
and reintroducing [rm,
(Vlf
F:-
Vd x
(10)
4
Eq. (9u), we obtain
- P(rf - ro)] 6
(11)
Thus the specific activity of the DNA is given by the difference of two terms. The first is the specific activity in the absence of RNA turnover and the second is a correction for t’he formation of less active DNA precursors from the RNA. In another experiment, the continuous administration of isotope was stopped, and the changes in activity of the nucleic acids were noted after an interval of time. We may assume that P = 0, since after cessation of administration the activity of the met’abolir CO? drops abruptly to a negligible value. In this case, Eq. (6) becomes rdR __ dt which on integration
= vz(A _ R) = f$
R 1
(12)
3
yields It = Roexp -
VII-,t (Vl + Vig)f=
or more exactly
1
R = R”[ l - 0: (V,+ i3;;: + V,) where R. is the initial Eq. (10) becomes
specific activity
in the RNA. Then for the DlSA,
d DO = A-VI = v, v3v4+ v3 x R. dt SubstJitutjing Eq. (13a) into Eq. (14) and integrating, UWf
(13a)
(14) we obtain
= @do + vl + ;; _ Tit (roRo - r&J
(15)
232
SWICK,
KOCH
AND
HANDA
which can be rearranged to give v4 A(Dd) -- A@6 = v, + vs - Vz’
(16)
Thus we have obtained equations predicting the isotope content of the nucleic acids at any time for these two different types of experiments in terms of reaction velocities and amounts of the component present. The values for these reaction velocities will be calculated from the experimental data to be presented. Usually, however, the results of metabolic studies are expressed either in terms of “half-lives” or turnover rates,4 the two being related by the fact that their product is 0.693 or In 2. Actually, the calculation of turnover rates has the advantage over the calculation of reaction velocities in that fewer experimental quantities are necessary. In the case of the nongrowing animal in “dynamic equilibrium,” only the data on the specific activity of a component is required for calculation of turnover rates. Even in the growing animal certain quantities having the dimensions of a turnover rate may be computed from the specific activity data and the extent of growth. For the present model three kinds of turnover rates may be obtained. The first is V,V,/(V, + V,)P, which gives the apparent rate of turnover of RNA. This quantity may be calculated from Eqs. (9) and (13). The other two quantities require a knowledge of the degree of growth. In the experiments reported here, the initial weight of the liver of the animal is estimated from the weights of the livers of animals of comparable age, and thus the degree of increase in liver weight is accurately known. The ratio of the final to initial liver weight (L) is given in Table I. If we assume that the increase in various components is proportional to L, then Eq. (16) becomes LDI - Do = v4 VI + vs Ro - LR
v, x a
(17)
and Eq. (11) becomes DL I.+1
_= d v4
v4
+ VI + vo -
vz PT
1 (18)
Thus from specific activity data and the factor of growth, the quantity [V4IWl + v3 - Vdl x (eo is obtained, and from this value Eq. (18)
XUCLEIC
233
ACID TURNOVER
yields VJd which is the rate of DNA synthesis. The quotient of Vdd divided by [ VJ( VI + VB - V,)] X (f/d) is (VI + VS - VJ/? or the third turnover rate. It should be noted, however, that in the case of “dynamic equilibrium” this becomes VI/r. Actually, [VJ(V, + VS- VZ)] X (f/d) seems to be the more interesting quant,ity because it, is related to the conversion of RNA label to DNA. The primary goal of the computation is to calculate these turnover rates. Then, secondarily, from estimates of the amounts of various components, individual reaction velocit,ies may be calculated for the various processes indicated in the scheme. If the growth rate was constant during the experiment,al period, the reaction velocities must have been constant also; but if deviation in growth rate has occurred, the calculated reaction velocGt,ies will be t’he average values. METHODS Animals were fed, at hourly intervals, purified diets containing CaC1”03 (Table I) (2) for periods ranging from 7 to 59 days, thus maintaining the radioactivity of the metabolic CO? at a constant level as established by the determination of t,he specific activity of the excret,ed urea. The nucleic acids were isolated by t,he proIf care is taken cedure described by Tyner et al. (5) with only slight modifications. to have all solutions at 0” before the addition of acid, the RNA fraction should be devoid of cont,amination with DNA. (A negative Dische test indicated less than 2% contamination.) In order to obtain DNA free of RNA, the DNA fraction was hydrolyzed to free bases and apurinic acid (6) by heating at 100” at pH 3 for 1 hr. Under these conditions the labile bond between the purines and deoxyribose phosphat,e is broken, while that between purines and any ribose phosphate present is only slightly affected as shown by the failure of these conditions to hydrolyze significant amounts of adenylic acid in control experiments. When the hydrolyzate is passed through a Dowex 2 Cl- column at a slightly acid pH, the nucleotides are retained, permitting the isolation of the free DNA purines. After separation of all the purines by cation exchange on Dowex 50 H+ (7), extraneous carbon compounds were eliminated by isolating adenine and guanine as the silver salts and then reconverting t,o the free purines. When adenine was degraded by heating with H&J1 to 190” for 1 hr. in a closed, C&free system under 60 cm. of NZ , I he as C6 position was liberated as CO* , the C-4 and C-5 cnrl)ons were obtained glycine, and C-2 and C-8 were converted to CO.5 The CO, was collected in liquid X;2 , and the glycine was isolated and decarboxylated wit’h ninhydrin to yield COZ Adenine and guanine were converted to CO2 by wgt combustion, and the specific radioactivity of all preparations was measured by proportional gas counting (8) with a standard deviation of ~1%. The weight of the liver at the beginning of the desired experimental period 5 Personal University.
communication
from
Dr.
G. Robert
Greenberg,
Western
Reserve
234
SWICK, KOCH AND HANDA
which was requisite for the estimation of growth, was obtained by sacrificing a number of rats of similar body weight and calculating a regression value for the rats used in the experiment. The standard error of the predicted value was calculated according to Tippett (9).
RESULTS
The results of the isotope analyses are present in Table I. Experiment A indicates that after an adult rat has been exposed to isotopic COz for 59 days the RNA guanine (and presumably adenine as well) has reached the theoretical maximum specific activity: 112 % of the urea value. On degradation of RNA adenine (Expt. C), more than 98 % of the radioactivity was accounted for in the C-6 and C-4 positions, as predicted, Of t.he isotope, 11% was in t,he C-4 position, which is derived from the carboxyl group of glycine, while 89 % of the total activity was in the C-6 position. Therefore, as the same distribution of activity must occur in the adult rat, the C-6 position has a specific activity of 89 % X 112 % = 100 % of the urea value. Therefore it may be concluded that COz is the sole precursor of the C-6 posit’ion; i.e., there is no dilution of the metabolic COa used for the synthesis of this position. As mentioned above, the specific activity of the 4-position is about 12 % of the metabolic CO2 , and this value is identical to that obtained for the carboxyl group of glycine from liver protein in this same animal (2). It can therefore be concluded that glycine is the sole precursor of the 4,5,7-positions of the purine because both the RNA and liver protein were at isotopic saturation in this animal.6 In Expt. A the specific activity of the DNA purines was slightly less than one-half of the specific activity of the RNA purines, indicating that 59 days is less than the apparent half-life of DNA. Experiments B, C, and D show that in growing rats the RNA purines are not isotopically saturated after 7-9 days exposure, and that considerable activity is associated with the DNA compounds. Since the liver weights have approximately doubled during the exposure period, one would expect the DNA to be approximately half-saturated if new growth and DNA synthesis are concomitant. This seems to be the case. Experiments D and E wereperformed to evaluate the conversion of adenine into guanine and their transfer from RNA to DNA. During the sec6 One may also conclude that RNA, like protein of rat liver (2)) consists entirely or almost entirely of substances that are being actively metabolized. It is of interest that the turnover rates of the two components are similar, although protein is present in much larger amounts.
Treatment
in liver
was calculated
in live
I
398 353
848 210
1068 91
118 53
1426 1439
-
1.87 2.22*
-
for the second period.
1.44a
I.610
8.9 1.3
-
1.2
10.3
71
94
65
122
236
129
316
318
394
2.30
181
176
-
403
326
2.20
1.07
84.5
RNA
in 100 counts/min./mmole
189
activity
212
168
Specific
in Liver
4denine
Acids
Adenine
6) Final wt. Initial
wrease
TABLE I and the Radioactivity of Nucleic
Constant exposure: 59 days; 2.2 rc./g. of amino acid diet (2) Constant exposure: 9 days; 5 pt./g. of 18% casein diet Constant exposure: 9 days; 5 pt./g. of 18% casein diet Constant exposure: 7 days; 16.8 pt./g. of 18% casein diet Constant exposure: 7 days; 16.8 pt./g. of 18% casein diet. No activity: 19 days; 12% casein diet Short exposure : 1.16 mc.; 120/, casein diet 21 days Short exposure: 1.16 mc.; 12% casein diet 21 days Short exposure: 1.16 mc.; 12% casein diet 21 days; 18% casein diet 21 days Short exposure: 1.16 mc.; 12% casein diet 21 days; 18% casein diet 21 days
0 Per cent increase
I. Weanling
H. Weanling
G. Weanling
P. Weanling
E. Weanling
D. Weanling
C. Weanling
B. Weanling
A. Adult
Animal
Growth
DNA
cpd.
65.4
43.8
103
238
333
278
128
-
81
236
SWICK,
KOCH
AND
HANDA
ond period (19 days), the specific activity of the RNA purines decreased sharply, in keeping with the known rapid turnover of RNA. That the specific activity of adenine decreased more rapidly than the specific activity of guanine is to be expected from the known ability of the rat to convert adenine into guanine but not the reverse; hence it appears that the radioactivity of RNA adenine is liberated by turnover and reincorporated into RNA guanine. The specific activity of the DNA purines did not fall to half of the original value during the second phase, as would have been predicted from the growth of the liver, indicating that RNA compounds contribute to the precursor pools from which DNA is synthesized. Each of the four rats of Expts. F-I was given 1.16 millicuries (me.) of NaHC1403 in three portions by intraperitoneal injection during the course of 1 day; two were sacrificed 21 days later; the other two, 42 days after the administration of radioactivity. In contrast to the reproducibility of the data obtained in the earlier experiments, there was a marked disagreement between similarly treated animals in the single-dose experiments. That the animals received identical amounts of Cl4 is evident from the similarity of the specific activities of the liver and muscle protein fractions of these animals (Table II). These discrepancies might result from variations between animals in the rates of renewal of RNA, or from variation in the rate of synthesis of nucleic acids, which are averaged out in constant exposure experiments but not in acute exposure studies. Our intention in the latter experiment was to measure changes in the radioactivity of DNA at a time when the contribution of radioactivity by RNA turnover would be minimal. During the first 21-day period the radioactivity of the RNA had decreased sufficiently to make only a small contribution to the DNA synthesized during the second phase, thus permitting a possible distinction between the dilution of DNA radioactivity by growth and any loss of DNA activity, for example, by turnover or cell death. In spite of the large variation between duplicate animals, there
Specific
TABLE II Activity of Tissue Proteins
Time si;Fyexposure, Experiment
F G H I
21 21 42 42
MUSCk,
Counts/min/mmole
4818 4916 1936 1570
C
Liver, counts/min/mmole
1177 1014 189 164
C
NUCLEIC
ACID
TABLE Apparent Experiment
l’wnover
III
Rate Constants
Equation No.
237
TURNOVER
of RNA
Adenine per da)
VI --.- X2 _____ VI + 1’0 r I Guanine per day
B
9
0.143
-
c I>
9 9
0.143 0.160 --
0.144 0.108
0.149
0.126
Av. D+E
13
0.129
0.074
F+H F+I G+H G+I
13 13 13 13
0.123 0.228 0.085 0.187
0.138 0.240 0.092 0.193
0.156
0.166
Av.
is some decrease in DNA specific activity solely by growth.
which cannot be accounted for
DISCCSSIOK
In Table III are shown the results of the esbimation of the apparent, rate of RNA turnover: VIVz/(Vl + I’,)?. This is only the net rate at which isotope enters or leaves RNA, and it includes the processes wherein RNA is broken down to yield fragments which are reutilized for RXA synthesis. The first group of estimates was obtained from experiments in which animals were exposed continuously to t,he isotope. The other estimates mere obtained from interanimal comparisons of t,he decrease in activit’y after administration of isotope had ceased, and, therefore, are less accurat,e. From these data it is apparent that the bulk of the Rn’A of liver is renewed at the same rate. At t,he end of the first 21-day period in Expts. F-I, the act’ivity of the RNA had decreased 25-fold. If even a small fraction of the RNA had a markedly smaller turnover rate (longer half-life) than the remainder of the RNA, the act,ivit,y in this hypothetical fraction would have decreased much less and the turnover measured during the next 21 days would he mainly of this component. Since the t\vo average estimates were the same, we may conclude that there is no such fraction. The presence of a small component, which is renewed more rapidly than the major port,ion of the RNA is, of course, not excluded.
238
SWICK,
KOCH
AND
HANDA
TABLE IV Puke
Composition
of Rat
Liver moles/g. liver
RNA DNA
Adenine Guanine Adenine Guanine
3.06 4.97 0.85 0.57
The other conclusion that may be drawn from these data is that intermediate adenine is a more efficient precursor of intermediate guanine than is guanine of adenine, in accordance with the observations of Brown et al. (10). This follows from the fact that the calculated turnover rates for adenine are higher than those for guanine in the continuous exposure experiments and lower in the decay experiments. This is to be expected if the reactions RNA adenine -+ intermediate adenine + intermediate guanine + RNA guanine are faster than the reverse reactions. We shall defer consideration of the significance of the quantitative estimate of VIVz/(Vl + V,)f to a later portion of the paper. The quantity V,/(V, + Vs - V,) is essentially the probability of feedback of an RNA purine molecule into the DNA. This quantity may be computed using Eq. (17), the data of Expts. D and E, and the ratio of purines present in these animals in the two types of nucleic acid. In Table IV are presented the amounts of the various purines found; each value represents the average of those determinations for which we were reasonably certain that the substances were obtained not only in radiochemically pure form, but also quantitatively. They are in substantial agreement with other values in the literature. There is considerably more RNA than DNA, and guanine predominates in the RNA, while adenine predominates in DNA. The absolute values fluctuate considerably, but the ratio between components was much less variable. Assuming that the ratio of RNA to DNA adenine is 3.6 and of guanine is 8.75, we obtain values for V,/(V, + V, - V,) of 12.1 and 13.45%, respectively. With these values one may then utilize Eq. (18) to calculate for growing rats the quantity VJd. This value should be relatively independent of inaccuracies in the estimates of material and also independent of the size of the animals; it is in fact the growth rate of liver DNA. Table V lists values of V,/d based on radioactivity measurements as well as those based on growth rate of liver (estimated from weight data). The estimates from the tracer data never exceed those obtained from liver weights; this fact leads to the following conclusions. First, the rate of
NUCLEIC
ACID
TABLE Estimate Expt. H
c D
V
of Growth
Rate [V.Jdl
From tracer data Adenine per day Guanine per day
0.07 0.056 0.062
239
TURNOVER
0.072 0.045
From liver weight per day
0.084 f 0.087 f 0.087 f
.004 .003 .012
cell death and DNA degradat’ion is insignificant compared with synbhesis in these growing rats. The est,imates of v.Ja for guanine are lower than for adenine, presumably for the same reason as they were for IISA. Scoondly, the hypothesis of Stevens et al. (11) and others (12), t,hat, when a cell divides both daughter cells receive only new DKA, is incorrect. This hypothesis requires that, under the conditions of our experiment in which the liver weights doubled (i.e., on the average, every cell divided once), there be a complete replacement of the DnTA; hence the rat,e of formation of DKA would have to be double the rate of growth of the liver. Since the rate of formation of DNA was similar to the rate of growt’h, we conclude, along with others (13, 14) that mit,osia is accompanied by the synthesis of only one cell complement of DNA. The growth rates estimated from tra.cer data are somewhat smaller thaii the mass growth rate. This discrepancy may be due to inadequacies of the model, although other models tested also yielded the same correction terms. On the other hand, it may be that the amount of DNA per unit weight is decreasing during the course of these experiments. Fukuda and Sibatani (15) have found that under the conditions of their experiments the DNA concentration is relatively caonstant in the age range used hew. However, in slightly younger animals they found a decrease in DXA concent’rat’ion of a magnitude which would account for the discrepancy in the present data. From 17Jd and VJ(V, + I’, - 1’2) X T,ld we may calculate the third turnover rate (Ii1 + I73 - V,)/?. From these three different turnover rates, t’he analytical data of Table IV, and the average rate of growt’h observed in these experiments, it is possible to calculate the individual reaction velocities V, through Ti6 for the nucleic acids of the growing rats. Average results for the young rats used in this study are tabulated in Table VI. Although in some instances the values are only approximate, hhey serve to indicat,e the relative magnitude of the processes involved. We have found that 4.9 kmoles of purine is synthesized and ‘L.-l pmoles is degraded in the liver of a 70-g. growing rat each day; 5.4 pmoles of purine
240
SWICK,
KOCH
AND
HANDA
.
TABLE Parameters
of Nucleic
VI
Acid Synthesis
Adenine, pm&s/day
2.6 1.8 1.0 0.22 1.6
VI
VP V3 V4 VS
in Young Rats” Guanine, pmoles/day
2.3 3.6 2.3 0.14 0.85
D It is assumed that the composition of the rat liver nucleic acid is that given in Table IV and that the average weight of the rat is about 70 g. (liver weight 3 g.).
is incorporated and 3.3 pmoles of purine is released from the RNA each day; 0.36 pmole of purine is incorporated into the DNA. Thus the rate of synthesis of new RNA exceeds the rate of de novo synthesis of purines. The expression of these results in terms of the concept of “half-life” would be inappropriate for two reasons. First, the apparent turnover rate of RNA, VIVJ(Vl + V,)P (Table III) is not only the rate of renewal of the nucleic acids but also includes the breakdown and resynthesis from fragments in the intermediary pools. The value of this quantity, in this case, corresponds to a half-life of about 4.6 days. This is comparable to previous estimates by Furst et al. (16), Bennett (17), and Bendich (18) for the adult rat. Secondly, two “half-lives” may be defined for every component in a growing animal. If one defines the half-life as that time required to synthesize at the observed rate an amount of RNA equal t’o 69.3% of that already present in the tissue, then the half-life is measured by 0.693r/Vz and is a considerably smaller quantity: 3.5 days as calculated from the present data. On the other hand, the time required to degrade at the observed rate an amount of the component equal to 69.3 % of that present in the tissue is of necessity longer in the growing animal. From the data presented here, this quantity is 6.4 days. Also, in the adult, nongrowing rat the use of the half-life concept for DNA turnover would be misleading. We have shown that DNA synthesis and cell growth
are concomitant
in the young
growing
rat, hence
it seems reasonable that DNA degradation is an aspect of cell death. We may interpret the DNA turnover as “cellular turnover,” with the probability of cell death either independent of its length of life, or dependent, with kinetics like those associated with the mortality of animal populations, normal red blood cells (19), or leucocytes (20). Although it is likely that both processes are involved,
the estimates
of the rate of replacement
of
DNA obtained in the two cases would differ by only 20 %. Selecting the second of the two alternatives, which assumes that no labeled DNA is de-
MJCLEIC
ACID
TURKOVER
241
graded during the experimental period, we may t’hen use Eq. (11) to calculate that the adult, nongrowing animal of Expt. A replaces 0.82 % of its DNA adenine and 0.82 % of its DNA guanine per day. In other words, slightly less than 1% of the DNA of cells of the rat liver is replaced daily: this estimat,e is of the same order as that calculated from the measurements of mit’otic activity by various workers (0.15-l % per day) (21,22), which suggests that intracellular turnover of DXA in living cells is either a much smaller or a non-existent process. It is well known that even the adult rat continues to grow very slowly; in this experiment, the extent of its growt,h (7 % in 59 days) account,s for 15 % of the DKA synthesized per day, leaving about 65 % of the DK:< formed per day for the replacement of dead cells. If all liver cells have the same life expectancy it could be inferred that the average life would l)e the reciprocal of this latter figure, or 150 days.’ These experiments are in complete accord with those recently presented by Fresco et al. (23). These authors administered adenine-XI5 and glycine-2-W to partially hepat,ectomixed rats and observed no appreciable loss of isotope from DSA between the first and fourt’h months after administrat,ion. Because regenerating rat liver was used, large amounts of radioactivit,y were incorporat’ed into all calassesof cells, but by the end of t,he first month the act,ivity in the RSA in all cells and in the DKA of the short-lived cells had been reduced. Since the cells that, contained radioactive DNA had all been produced veryshortlyaft’erhepatwt,omy, it may be concluded from their data, and in accord with our findings, that) the average life span of hepa,tic ~11s is great’er t’han 4 months.
The incorporation of CY402into the nucleic acids of rat liver has been st’udied by using the technique of cont’inuous administration of isotope. i The 150-day life span clearly represents an average for all liver cells. Presumably the life span of the hepatic cell is somewhat longer than 150 days and those of some of the other cell types may be considerably shorter. As mentioned above, 1here is significant loss of radioactivity from the DNA between the 3rd and the 6t.h week following the short exposure to C”O, . After correction is made for the small contribution of RNA to DNA in Expts. P-I, it may be calculated that very roughly 50% of the DNA activity present at the end of the 3rd week had disappeared by the end of the 6th week. Since the incorporation of isotope in the various types of cells should be inversely proportional to the life span of the class, one would infer that approximately 10% of the cells in the liver had a life span bet,ween 3 and 6 weeks.
242
SWICK,
KOCH
AND
HAND.4
The following conclusions were drawn: 1. Metabolic CO2 enters the C-6 position of the purines without dilution. Glycine enters the C-4, C-5, and N-7 positions, also without dilution. 2. In the young growing rat the percentage of new liver DNA synthesized per day is similar to the percentage increase in liver weight. Thus the synthesis of only one cell complement of DNA is associated with each mitosis, and the existing DNA is retained. 3. In young, rapidly growing rats, the rat,e of RNA renewal is rapid and all of the RNA is renewed at the same rate. 4. In the adult rat liver, the average life span of the DNA molecule is estimated to be at least 150 days. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23
BUCHANAN, D. L., J. Gen. Physiol. 34, 737 (1951). SWICK, R. W., AND HANDA, D. T., J. Biol. Chem. 218,577 (1956). DANCIS, J., AND BALIS, M. E., J. Biol. Chem. 207,367 (1954). BENNETT, E. L., AND KRUECKEL, B. J., Biochim. et Biophys. Acta 17,503 (1955); 17, 515 (1955). TYNER, E. P., HEIDELBERGER. C., AND LEPAGE, G. A., Cancer Research 13, 186 (1953). TAMM, C., HODES, M. E., AND CHARGAFF, E., J. Biol. Chem. 196, 49 (1952). HURLBERT, R. B., AND POTTER, V. R., J. Biol. Chem. 196,257 (1952). BUCHANAN, D. L., AND NAKAO, A., J. Am. Chem. Sot. 74, 2389 (1952). TIPPETT, L. H. C., “The Methods of Statistics,” p. 265. John Wiley and Sons, New York, 1952. BROWN, G. B., ROLL, P. M., PLENTL, A. A., AND CAVALIERI, L. F., J. Biol. Chem. 1’73, 469 (1948). STEVENS, C. E., DAOUST, R., ASD LEBLOND, C. P., J. Biol. Chem. 20% 177 (1953). Indicators,” p. 325. Interscience Publishers, Inc., HEVESY, G., “Radioactive New York, 1948. BARTON, A. D., Federation Proc. 13, 422 (1954). KIHARA, H. K., AND SIBATANI, A., Biochim. et Biophys. Actn 17, 579 (1955). FIJHUDA, M., AND SIBATANI, A., J. Biochem. (Japan) 40, 95 (1953). FURST, S. S., ROLL, P. M., AND BROWN, G. B., J. Biol. Chem. 183, 251 (1950) BENNETT, F,. L., Biochim. et Biophys. Acta 11, 487 (1953). BENDICH, A., Exptl. Cell Research, Suppl. 2, 181 (1952). LONDON, I. M., SIIEMIN, D., WEST, R., AND RITTENBERC, D., .I. Biol. Chem. 179, 463 (1949). KLINE, D. L., AND CLIFFTON, E. E., Science 116,9 (1952). BRUES, A. M., AND MARBLE, B. B., J. Exptl. Med. 66, 16 (1937). WIDNER, W. R., STORER, J. B., AND LCSHBAUQH, C. C., Cancer Research 11, 877 (1961). FRESCO, J. R., BENDICH, A., AND RUSSELL, P. J., JR., Federation Proc. 14,214 (1955).