- Email: [email protected]
Metabolic activity and intracellular distribution of nucleic acid phosphorus in regenerating liver
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
67,
350-365 (19%‘)
Metabolic Activity and Intracellular Distribution of Nucleic Acid Phosphorus in Regenerating Liver1 Christine D. Jardetzky2 and Cyrus P. Barnum From the Department
of Physiological Chemistry, Medical Minneapolis, Minnesota
School,
Received July 9, 1956 INTRODUCTION
Differences in the intracellular distribution and metabolic activity of ribonucleic acid phosphorus (RNAP) of various cell fractions from normal adult mouse liver and mammary carcinoma have recently become apparent (l-3). In the caseof the tumor tissue the specific activity data have been consistent with a metabolic scheme involving the different cell RNA’s, the deoxyribonucleic acid (DNA), and an unknown immediate precursor pool. Based on this metabolic scheme, on the calculated specific activity-time curve of the unknown precursor, and on the assumptions of the steady state and metabolic homogeneity of the various nucleic acid phosphorus pools, the fraction that is renewed per hour was estimated. This was found to be 118,596, and 150%/hr. for the cytoplasmic particulate and supernatant RNA’s, and nuclear RNA, respectively (2). Other values reported in the literature or calculated from half-lives, based on the loss of radioactivity from the particular nucleic acid and on the assumption that the specific activity of the immediate precursor is zero, (l/(rate/total amount) = tl,Jln 2) are 1.72 for total rat liver RNA (4), 0.52 for regenerating rat liver total RNA purines (5), 0.31 for total RNA purines in liver from tumor-bearing rats (6), and 5.9 and 2.7%/hr., respectively, for nuclear RNA and cytoplasmic RNA adenine from adult mouseliver (7). The marked differences in the fraction of RNA that is renewed per hour may be partly 1 These investigations were supported by a grant-in-aid from the American Cancer Society. * Present address: Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena 4, California. 350
NUCLEIC
ACID PHOSPHORUS IN LIVER
351
due to the additional assumption in Refs. (5-7) that the specific activity of the immediate precursor was zero at the time during which the loss of radioactivity from the particular nucleic acid fraction occurred. For the purpose of comparing the nucleic acid metabolism of normally growing tissue to that of either normal adult liver or tumor tissue, regenerating liver was chosen for study. The results from a study of distribution of RNA in different cell fractions obtained by differential centrifugation 60 hr. after partial hepatectomy and those from a shortterm Ps2incorporation into the nucleic acids are reported in this communication. A mathematical analysis of the data in an attempt to determine possible precursor-product relationships and the calculation of minimum values for the per cent renewal of the various RNA’s are also included. Such limiting values are felt to be more realistic than those obtained on the assumption that the immediate precursor has zero specific activity, but at the same time are not subject to the hazard of trying to approximate the specific activity-time curve of an unknown precursor. EXPERIMENTAL Male ZBC mice3 between 1.5 and 2.5 months of age were partially hepatectomized as previously described (8). They were housed in groups of 5-10 in wooden boxes and given Purina Fox Chow ad libitum and 10% dextrose for drinking until the time of sacrifice. The animals were operated on always between 9 and 11 p.m. and were sacrificed between 9 a.m. and 2 p.m., 0.5-6 hr. after the injection of P3*, and 58-64 hr. after partial hepatectomy. The P3* solution received from the Oak Ridge National Laboratory was diluted with 0.857o NaCl, and an amount of 2 microcuries bc.)/O.Ol ml./g. body weight was administered intraperitoneally. Pools of livers from 3 to 4 mice were homogenized in alkalinized saline in a Potter-Elvehjem homogenizer. The final volume of the homogenate was 15 ml. An aliquot was taken for the determination of the inorganic phosphorus (IP) specific activity. The rest of the homogenate was then fractionated by differential centrifugation into the following fractions: nuclear (N) at 1400 X g for 5 min. (or at 23,000 X g for 5 min. when the mitochondrial fraction was not saved), mitochondrial (L) at 23,000 X g for 5 min., microsomal (M) at 23,000 X g for 1.5 hr., ultramicrosomal (U) at 100,000 X g for 1 hr., and supernatant (8).
The N pellet was further purified by repeated washings, 12-16, with cold 24/, citric acid followed by two quick washings with cold alkalinized saline (9). The 3 We are indebted experiments.
to Dr. J. J. Bittner
for supplying
the mice used in these
352
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
phospholipides were then extracted with 95% ethanol followed by treatment with hot Bloor’s reagent and an ethanol wash. These extracts were combined and evaporated to dryness, and the phospholipides were picked up in petroleum ether. The lipide-free nuclei were treated with cold 0.1 M bicarbonate-carbonate buffer, pH 10, which removed about 50% of the total RNA (9). This fraction of N-RNA is labeled N-RNA1 . It was further purified from traces of acid-soluble phosphorus (ASP) and DNAP by precipitation with an equal volume of cold 10% trichloroacetic acid (TCA), hydrolysis of RNA with 0.1 M NaOH, and a second precipitation with cold 10% TCA. The DNA of the N pellet, after N-RNA1 removal, was solubilized at 100°C. with 6% NaCl buffered at pH 10 and then precipitated with 2 vol. of ethanol. The DNA in this sediment was solubilized in hot 10% NaCl, reprecipitated with ethanol, and further purified by a Schmidt-Thannhauser extraction as previously described (9). In some cases the ethanoldyc NaCl-pH 10 supernatant containing the other half of the total N-RNA was saved, evaporated to dryness, and extracted with cold 5% TCA. This was called the N-RNA2 fraction.
Cytoplasm The tubes containing the M and U pellets were rinsed once with cold alkalinized saline. The neutral fat adhering to the sides of the tubes was removed with gauze, and the pellets were transferred quantitatively into centrifuge tubes using 10% NaCl. Solid NaCl was added to the S fraction to give a final concentration of 10%. The samples were then placed in a water bath at 100” to solubilize the RNA and denature the protein. The RNA in the 10% NaCl extracts was precipitated with 2 vol. of ethanol, re-extracted with hot 10% NaCI, and reprecipitated with ethanol, solubilized in cold distilled water, precipitated with cold 10% TCA, and finally hydrolyzed with 5% TCA as previously described (3). However, difficulty was encountered in precipitating, with cold TCA, the RNA that was dissolved in water. Therefore, in some experiments this step was substituted by washing the second alcoho1 precipitate with cold 5% TCA, hydrolyzing for 30 min. at 80-65” with 0.1 M NaOH, cooling, and precipitating the glycogen and traces of contaminating protein with cold ethanol-TCA mixture to give a final TCA and ethanol concentration of 5 and SS’%, respectively. The RNA purified according to the altered procedure had the same specific activity as that obtained from the older method except at the 0.5-hr. period with Paz when it was almost doubIed. The older method was used in all cases in purifying RNA for specific activity determinations at 0.5 hr. The phospholipides were extracted from the sediments of the different cytoplasmic fractions after the removal of RNA, and were purified as described under Nuclei. The protein-P fractions were obtained from the purified N pellet and M, U, and S fractions after the complete removal of lipides and nucleic acids (3). The radioactive samples and standards were prepared for counting and were counted with a Geiger-Miiller dipping tube as indicated previously (3). Phosphorus was determined by the method of Fiske and SubbaRow (10) on the same aliquotsused for counting. Specific activities are reported as counts/min./mmole P
NUCLEIC
ACID PHOSPHORUS
353
IN LIVER
as per cent of counts/min. injected/g. body weight (BCC).’ Relative specific activity refers to counts/min./~g. P as per cent of counts/min./~g. IP. For the quantitative estimation of nucleic acids, the constituents of the different cell fractions were precipitated and washed with cold 5% TCA, and hydrolyzed with hot TCA (1). An aliquot from whole homogenate was treated similarly. DNA and RNA were determined by the diphenylamine and orcinol-HCI reactions (1). The absorbancy of RNA solutions was corrected for the presence of DNA and glucose whenever necessary.6 These corrections never amounted to more than 5-10% of the absorbancy of the solutions. RESULTS
From Table I it can be seenthat about 43 % of the total cell RNA is associated with the microsome fraction. Approximately RNA can be accounted for. The incomplete recovery
70 % of the total is probably due to
loss of whole cells that escaped breakage in the process of homogenization and perhaps due to some hydrolysis of RNA into components during the period of sedimentation of the
acid-soluble various cell
fractions. The acid-soluble constituents of the various pellets are probably contaminated by those of the supernatant liquid trapped in the pellets. A large amount of the total cell phospholipide probably sediments at 1400 X g for 5 min. since only about 50% could be recovered from
the different
fractions.
It is interesting
to note that
the RNA
to
lipide-P ratio of the N pellet is approximately the same as that of the M pellet, which is about one-half that of the U and S fractions, and five times
that
of the L pellet.
The
amount
of IP/g.
tissue was found
to be
226 f 9 pg., and approximately 26 % of the total ASP. The specific activities of IP and the different nucleic acids are seenin Table II. Not shown in this table are the N-RNA* specific activities at 0.5, 1, and 2 hr. after Ps2injection; these are 1423 f 115, 1940 f 256, and 2834 f 724, respectively. Comparison of N-RNA1 and N-RNA, does not reveal any appreciable differences in their mean specific activity, although
each may be a mixture
of metabolically
distinct
RNA’a.
Also,
the specific activity of L-RNA at 0.5 and 1 hr. was almost identical with the mean specific activity of M-RNA at those times. The specific activity-time curves of the IP and the various nucleic acids are seen in Figs. 1 and 2. It is noted in Fig. 2 that the specific activity-time curve for DNA has a very small slope between 3 and 6 4 The use of the Biological Concentration mended by Schulman and Falkenheim (11). 6 C. P. Barnum, unpublished experiments.
Coefficient
(BCC) has been recom-
354
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
hr. This observation suggested that the rate of entrance of IP into the DNA precursors and/or the rate of DNA synthesis from these precursors might be decreased during the later interval of the kinetic study. Therefore, the 1-hr. incorporation of Pa2 into the DNA and other constituents was studied as a function of the time after the operation and the time of day when the animals were sacrificed. The results are given in Table III. It is seen that the relative specific activity of DNA is higher in the TABLE I Regenerating Liver Composition 60 Hours after Partial Hepatectomy Average values are based on 4-10 duplicate analyses. For each analysis pools of livers from 3 to 4 mice were used. RNA and DNA were determined with the orcinol-HCl and diphenylamine reactions. Micrograms/g. Cell fraction0
H
ASP
fresh liver RNA
f optimal Lipid&
estimateb RNA Lipid&
10,074 1,201 8.4 f406 f138 N 424 23 18.2 f63 f5 N= 590 32 L 23 390 2.9 135 f4 f50 f22 LC 40 668 231 M 15.2 62 4,340 286 f19 f541 f64 U 10 662 19 35.6 f3 f20 f4 S 720 919 30 31.0 f39 f34 f6 NC, Le, M, TJ, S 833 7,179 598 a H denotes homogenate; N, nuclei; L, mitochondria; M, microsomes; U, ultramicrosomes; S, supernatant; ASP, acid-soluble phosphorus. z(r - zp bf N-1. d c Constituents corrected for the loss of nuclei or mitochondria. The correction factor for nuclei was based on the quantitative estimation of DNA from H (2012 f 154agJg.) and that from N (1447 f 202pgJg.). An approximate correction factor for mitochondria was based on the RNA to lipide-P ratios of H (8.4), L (2.9), and of the combined citric acid washes of the N pellet (6.9). A preferential loss of mitochondria could decrease this ratio from 8.4 to 6.9, and the difference between the total amount of lipide-P (521 pg./g.) in the combined citric washes and 6.9/8.4 times this amount was attributed to the mitochondria. The RNA was 2.9 times the amount of iiplde-P. 879 3279
NUCLEIC
ACID
PHOSPHORUS
IN
TABLE II Nucleic Acid and IP Specijic Activities BCCb Hours
f
optimal
355
LIVER
in Regenerating
Livera
estimate
with Pa’
0.5
IP
DNA
15,155
19 f2 49 ZIG3
N-RNA1
M-RNA
U-RNA
S-RNA
19 51 fl,lSl fl f2 f12 1 11,841 32 73 144 fl,O90 f6 f21 f35 2 6,541 114 112 192 251 f859 f38 f13 *30 f22 3 4,679 167 230 310 408 *377 f24 zk.33 f32 f84 4 4,170 180 2,414 292 407 465 f128 f32 zk283 f22 f47 f26 6 3,068 200 2,041 389 481 545 f105 f21 f102 f28 f21 f47 a The mice were sacrificed 58-64 hr. postoperatively from 9 a.m. to 2 p.m. at times indicated after P32 injection. Pools of 3-4 livers were used for each analysis, and a total of 4-10 analyses per time point. b BCC = counts/min./mmole P as per cent of counts/min. administered/g. body weight.
0
I
1,120 zk67 2,075 f275 2,566 f228 2,583 f242
2 3 Time anhours4
6
5
6
FIG. 1. Specific activity-time curve for inorganic phosphorus (IP), nuclear (N) RNA, supematant (S) RNA, and DNA of mouse liver 58-64 hr. after partial hepatectomy. Vertical lines in this and each of the subsequent figures indicate f one standard error of the means. Biological Concentration Coefficient (BCC) refers to counts/min./mmole P as per cent of counts/min. injected/g. body weight.
356
CHRISTINE
D.
600
JARDETZKY
,
AND
CYRUS
P. BARNUM
t
I
500 !&!400 Cx
-
3 4 5 6 Time in hours FIG. 2. Specific activity-time curves for three cytoplasmic RNA’s, supernatant (S), ultramicrosomal (U), and microsomal (M) RNA and DNA of mouse liver 58-64 hr. after partial hepatectomy. 0
I
2
than in the early afternoon, although the difference was not found to be statistically significant (P = 0.1). More recent results on the incorporation of Pa2into the DNA of young ad l&turn fed mice show a threefold decrease in the 2-hr. relative specific activity of DNA at 8:30 p.m. compared with that at 4:30 a.m. (12). These results on immature growing livers indicate that between 8 a.m.
morning
One-Hour Time of sacri6ce 1 hr. with P*’
TABLE III Relative Specific Activity (RSA)= of Regenerating Liver Constituents at Different Times of Day RSA -I optimal estimate chntsjmhL/fJg. z;,B’
- ASPC
N-RNA1
IP
DNA
0.48 71 16.4 9:30 a.m., 69 hr. after 8 12.9 ~2.7 zkO.11 partial hepstectomy 70 15.3d 0.35 1:30 p.m., 65 hr. after 8 f3.2 ho.5 10.06 partial hepatectomy a RSA = counts/min./pg. P as per cent of counts/min./fig. * Pools of two livers were used for each analysis. = This is only the organic fraction of the ASP. d Average of three analyses.
2,350 1117 2,224 f213 IP.
P
NUCLEIC
ACID
PHOSPHORUS
IN
LIVER
357
and 2 p.m. one would expect about the decrease in Pa2 incorporation into DNA that is noted in Table III for the regenerating liver. In Tables IV and V the specific activities of the various phospholipides and protein-P fractions are seen. Their specific activities are of the same order of magnitude as those of N-RNA. It may be noted also that the nuclear lipide-P and protein-P fractions have a lower specific activity at all times than that of any of the cytoplasmic fractions. DISCUSSION
The distribution of nucleic acids in regenerating liver 60 hr. after partial hepatectomy is more like that of normal adult liver than mammary carcinoma (1, 2). Most of the RNA is present in the microsome fraction in the adult and 60-hr. regenerating liver, while in tumor a relatively high percentage of the total cell RNA (24) is found in the ultramicrosome fraction, and it is indistinguishable in its specific activity from the RNA of microsomes. The microsome fraction is thought to be composed of RNA-rich particles associated with cytoplasmic membrane fragments (13-15). It appears then, that there is a greater number of such particles freely dispersed in tumor than in adult or in 60-hr. regenerating liver cells, and that the RNA from the free cytoplasmic particles in tumor is in rapid equilibrium with that of the particles attached to the endoplasmic reticulum, because of their similar specific activities. Howatson and Ham (16) predicted from electron microscope studies that a smaller percentage of the total cell RNA would be found in the microsomes from liver tumor than from normal liver cells. Their prediction was based on the observation that the cytoplasm of liver tumors had fewer reticular structures and a greater number of freely dispersed particles than that of normal liver. Ultracentrifugal and electrophoretic analyses carried out by Petermann et al. (17) on cytoplasmic supernatants after removal of nuclei and mitochondria have shown a decrease in the total amount of microsomes and in a heavy “B” component, along with an elevation in the amounts of the lighter “C” and “E” components 2 days after partial hepatectomy in rats. The amounts of the latter components were back to normal levels 3 days after the operation. The shifts in the amounts of RNA associated with the various cell fractions noted under different physiological and pathological conditions suggest that certain metabolic precursor-product * In certain instances in the literature the term “microsome” encompasses both the ultramicrosomal and the microsomal fractions described here.
358
CHRISTINE
D. JARDETZKY TABLE
Specific
Activity
of Phospholipide of Regenerating
AND
N
M
0.5
824
963 f147 1,984 f373 2,759 +471 2,904 f243 3,122 1138 2,875 f355
2 3
2,184c 1200 2,604
4 6
2,465 f87
P. BARNUM
IV from Different Cell Fractions Liver0 BCC f optimal estimate
Hours with Pa*
1
CYRUS
U
s
1,031
1,003
1,897* f12
1,753* *3 2,427
2,639 f202 2,957
2,436 f201 2,855
2,856* *51
2,816* 4x54
a See Table II. * Average of two analyses. c Average of three analyses. TABLE Specific
Activity
Hours with Paz 0.5 1 2 3 4 6
V
of Protein-P from Different Cell Fractions of Regenerating Livers BCC * optimal estimate N 1,315 ~328 1,611 f243 1,150 f333 906 1,727 *293 1,226” f598
M 1,892 f216 2,386 f224 2,438 zt348 2,479 2,941 *350 2,553” f131
S 2,773* f322 3,44O* f300 2,654 3~664 2,701 2,454 f102 2,166” f154
Q See Table II. * Average of two analyses. c Average of three analyses.
relationships may be operating and should be the subject for further study. The rationale for carrying out the kinetic study between 58 and 64 hr. after partial hepatectomy was that the growth rate of the liver at
NUCLEIC
ACID
PHOSPHORUS
IN
359
LIVER
that time appeared to be less variable than at earlier times after the operation. This was based on the values of the 1-hr. relative specific activity of DNA which showed a peak at 36 hr. but remained fairly constant between 54 and 72 hr. postoperatively (8). Therefore, it seemed reasonable to assume that during that time (a) the various liver constituents were synthesized at constant rates, and (b) the net increase in the total amount of these substances during the experimental period was very small. These two assumptions imply that the metabolic system is very close to the steady state. In a separate communication (18), an equation of first order in specific activities is derived from which the rate of appearance of a polymer from a homogeneously labeled precursor may be calculated. It is also shown that for any quantitative mathematical treatment of the results, the following assumptions must be valid: 1) conditions (a) and (b) above, and Z?) absence of compartments which form a barrier for the complete and instantaneous mixing of a specific nucleic acid pool, for example, the N-RPr’A pool. Cell membranes in the liver do not allow mixing of the RN-4 of one cell with that of another. Therefore, if the mathematical treatment is to be valid, it must be assumed that the reactions from precursor to a specific RNA are identical in all cells. With these assumptions in mind several schemes were tested for agreement with the experimental data, some of which are shown below. A F? N-RNA
F? S-RNB
B F? M-RNA
e
S-RNA Q U-RNA
G? M-RNA
--+
DNA t
S-RNA e
X F’t U-RNA
M M-RNA I
--+
T G X F), N-RNA n S-RNA F1 U-RNA
*
M-RNA
SchemesA and C are patterned after Jeener’s earlier and later suggestions according to which N-RNA is the immediate precursor of S-RNA which in turn is the immediate precursor of particulate RNA (19) on one hand, and, on the other, the particulate and nonparticulate RNA’s are formed independently of one another (20). Scheme B depicts Chantrenne’s hypothesis that larger cytoplasmic particles are formed from smaller ones (21), and schemeD is almost identical with that suggested by Barnum et al. (2) for the nucleic acids from mammary carcinoma.
360
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
None of these schemes (nor several variations of them) were found to satisfy the data from regenerating liver. The only relationship that was in reasonable agreement with the data is the following one: F? IP 4
The specific-activity are
N-RNA
4
M-RNA
equations applying to this system in the steady state dM = z (N - M) dt
and
dN = 2 (I - N) + 2 dt Equations
(1) and (2) in the integrated
N = z(l
(M - N) form are
- Y) + vf (/.l - v)
where capital letters represent specific activities, Greek letters represent the areas under the specific activity-time curves estimated with the use of Simpson’s rule (2), the V’S represent rates, and n and m the amounts of N-RNA and M-RNA/g. fresh liver. The values of the constant vz/m obtained from Eq. (3) at 0.5, 1, 2, 3, 4, and 6 hr. after Pa2 injection are 0.0322, 0.0295, 0.0344, 0.0378, 0.0366, and 0.0334, respectively, with a mean value of 0.034. According to this scheme the microsomal RNA is formed from nuclear RNA at the rate of 3.4% per hour. In Fig. 3, which is a graphical solution of Eq. (4), it is seen that a fairly good straight line goes through the points. The constants vi/n and (Q/n are equal to 0.24 and 0.34, respectively, and are obtained from the intercept and the slope of the line described by Eq. (4). These results imply that the nuclear RNA is formed from IP and is diluted by M-RNA at the rates of 24 and 34 %/l-u-., respectively. Since the fractions v2/m and (ve2)/n were evaluated independently from each other from Eqs. (3) and (4), and if it is assumed that the ratio v~/(v-2) is close to
NUCLEIC
ACID
,
0
PHOSPHORUS
I
IO FIG. 3. Graphical
1
IN
361
LIVER
4
20
I
30
g! solution of Eq. (4).
unity, the ratio of the amounts of M- and N-RNA, m/n, must be at least equal to 10. From Table I it is seen that m/n = 7.4, which is in pretty good agreement with the value predicted from the metabolic scheme. However, these quantitative metabolic relationships must be accepted with reservation because of the assumptions implied in the mathematical analysis. In partial agreement with this metabolic scheme is the experimental evidence presented by Goldstein and Plaut (22) that the nuclear RNA is the precursor of cytoplasmic RNA but that this is an irreversible processin ameba cells. In an attempt to avoid making the assumption of homogeneity of a particular nucleic acid fraction and guessingat the immediate precursor, the specific activity-time curve of the liver inorganic phosphorus was used to obtain minimum values for the fraction that appears per hour, as shown in the previous communication (18). These minimum values for the various RNA’s are underlined in Table VI. It is interesting to note that a minimum value for the renewal rate of N-RNAP is 25 %/hr. This value is lower than that obtained from the metabolic schemeaccording to which nuclear RNA derives its phosphorus from the inorganic phosphorus and the microsomal RNA with the over-all rate of 58 %/hr., but it is higher than most values reported in the literature for either
362
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
TABLE VI Approzimate Fractional Rates of Appearance of Various Ribonucleic Acid Phosphorus Fractionsa Fractional Time
with
Pap
N-RNA
rate/k
S-RNA
X 100 U-RNA
M-RNA
1.16 0.42 0.13 19.5 1.20 0.60 0.27 15.1 1.39 0.95 0.57 13.0 1.54 1.22 0.86 11.1 1.67 1.32 0.99 3.4 1.54 1.36 1.09 = Derived by substituting the area under the true immediate precursor specific activity-time curve by that of the IP of the liver (18). Minimum values for the fraction of nucleic acid phosphorus appearing per hour are the italicized maximum values. The assumptions of an essentially homogeneous IP pool in the liver and the steady state within the 6-hr. interval 53-64 hr. after partial hepatectomy are implied. 0%
24.7
1 2 3 4 6
total or nuclear RNA (4-7). For the cytoplasmic RNA’s the minimum values range from 1 to 2%/hr. The fact that U-RNA has a specific activity-time curve intermediate between those of M and S-RNA suggestedthe possibility that it may be a mixture of particles containing S and M RNA. In order to determine how much S and M RNA would give a mixture whose specific activitytime curve was similar to that found experimentally for U-RNA, the following equation was set up UU = u,S + u,,,M
(5)
where u is the amount of U-RNA/g. liver, us and u, are the amounts of U-RNA from the S and M RNA pools, respectively, and the capital letters are the observed specific activities of the RNA’s. Since u = us + U Vlk, Eq. (5) can be reduced to the following one with only ua as an unknown. lb=
u(V - M) S-M
(6)
From the amount of U-RNA (u = 662 pg./g. liver, Table I) and Eq. (6), us was calculated and was found to have the following values at 0.5, 1, 2, 3,4, and 6 hr.: 194, 242, 381, 299,442, and 389 with an average of 325 pg./g. Then, by difference, the average u,,, is 337 pg./g. Using the
NUCLEIC
ACID
PHOSPHORUS
TABLE
IN
VII
Observed and Calculated SpeciJic Activities Hours with P32 Observed BC0 Calculated
BCC”
= From Table II. b Obtained from Eq. (5) mixture of M- and S-RNA.
0.5 19 f2 28
1 73 f21 87
on the
basis
363
LIVER
2 192 zt30 180 that
U-RKA
(BCC) of U-RNA 3 310 f32 317
4 407 f47 377
is approximately
6 481 f21 466 a 50:50
average values of us and u, , the specific activity of U-RNA was calculated from Eq. (5). The calculated and observed values are given in Table VII. It is seenthat with the exception of the 0.5-hr. point, all the rest of the points fall within the range of f one optimal estimate of the mean. More experimental work, however, is necessary before one can conclude that the U fraction is composed of particles the RNA of which is a 50:50 mixture of S- and M-RNA. It is pertinent to mention that contamination of U from S-RNA due to the trapping of a small volume of supernatant fluid in the U pellet does not amount to more than 3 % of the total U-RNA. With regard to the DNA, it is seen from Table III that the 1-hr. relative specific activity may be lower in the early afternoon than in the morning. This is in agreement with recent results that establish a marked daily variation in the 2-hr. relative specific activity of the DNA and mitosis in the immature growing liver (12). Such a daily variation also affects the mitotic index and P32incorporation into the DNA of regenerating mouse liver (12). The morning-afternoon changes in the l-hr. relative specific activity of DNA (Table III), as well as the flatness in the DNA specific activity-time curve between 3 and 6 hr. after Ps2injection (Fig. 2), imply that the rate of DNA formation from its immediate precursor may not be constant during the 6-hr. interval of the kinetic study, since there is no change in the 1-hr. specific activity of either the IP or the N-RNAP. For this reason, it did not appear justifiable to assume the steady state for the rates of reactions leading to DNA synthesis, and metabolic schemesinvolving the DNA were not tested. The hypothesis, proposed originally by Ahlstrijm et al. (23), and later by Barnum et al. (2) and Stevens et al. (24), that the DNAP of growing cells may be metabolically unstable, remains to be tested in systems in which DNA synthesis proceeds at a constant rate and the specific activity-time curve of the immediate precursor is known. It should be
364
CHRISTINE
D. JARDETZKY
AND CYRUS P. BARNUM
mentioned that, in contrast to the decreasing trend in the 2-hr. relative specific activity of DNA between 8:30 a.m. and 4;30 p.m., the various RNA’s during this time exhibit fairly constant relative specific activities in the immature growing liver (12) Y ACKNOWLEDGMENTS The authors gratefully acknowledge the skillful assistance of Miss Marlys J,. Hawkinson and Mrs. Margaret M. Scheller in the many chemical and radioactive
analysesreported. SUMMARY
The specific activity-time curves for the various liver nucleic acids were determined within a 6-hr. period from 58 to 64 hr. after partial hepatectomy in mice using Paz as the tracer. The nuclear, supernatant, ultramicrosomal, and microsomal RNA’s are listed in the order of decreasing specific activities. The protein and phospholipide phosphorus specific activities increased rapidly during the first hour, and all, with the exception of those from nuclei that were lower, were almost equal to the IP specific activity 6 hr. after Pa2injection. A study of the quantitative distribution of RNA revealed that about 40% of the cell RNA in the 60-hr. regenerating liver is associated with the microsome fraction. Only about 5-10% could be accounted for by each of the RNA’s of the other fractions. Assuming the condition of the steady state and metabolic homogeneity of the various nucleic acid phosphorus pools, equations representing different metabolic schemes were tested. It was shown that the data were in reasonable agreement with a metabolic scheme according to which M-RNA is formed from N-RNA at the rate of 3.4%/hr., and N-RNA appears at the rate of 24 and 34%/hr. from IP and M-RNA, respectively. Limiting fractional rates for the appearance of the various RNA’s were calculated using the liver inorganic phosphorus specific activity-time curve in place of that of the immediate precursor; these were l-2 % and 25 %/hr. for cytoplasmic and nuclear RNA, respectively. This fractional renewal rate for N-RNA is much greater than most values reported in the literature and, even so, must be considered a limiting minimum value. It seemedunjustified to assumethe steady-state condition during the kinetic study for the rates of reactions leading to DNA on the basis of 1 Unpublished
results.
NUCLEIC
ACID
PHOSPHORUS
IN
LIVER
365
the shape of its specific activity-time curve and on the morning-afternoon differences in the 1-hr. relative specific activity. Therefore, metabolic reactions involving DNA were not tested. An equation was developed to test whether the U fraction is a mixture of particles containing M- and S-RNA. The calculated specific activities from approximately a 50:50 mixture of S- and M-RNA were in fairly good agreement with the observed values of U-RNA. REFERENCES 1. HUSEBY, 2. BARNUM,
R. A., AND BARNUM, C. P., Arch. Biochem. 26, 187 (1950). C. P., HUSEBY, R. A., AND VERMUND, H., Cancer Research
13, 880
(1963). 3. 4. 5. 6.
BARNUM, C. P., AND HUSEBY, R. A., Arch. Biochem. 29, 7 (1950). HAMMARSTEN, E., AND HEVESY, G., Acta Physiol. Stand. 11, 335 (1946). FURST, S. S., ROLL, P. M., AND BROWN, B. G., J. Biol. Chem. 183, 251 (1950). TYNER, E. P., HEIDELBERQER, C., AND LEPAGE, G. A., Cancer Research 12,
158 (1952). R., AND MARSHAK, A., J. Biol. Chem. 206, 585 (1963). C. D., BARNUM, C. P., AND VERMUND, H., J. Biol. Chem. 222, 421 (1956). BARNUM, C. P., NASH, C. W., JENNINGS, E., NY~AARD, O., AND VERMUND, H., Arch. Biochem. 26, 376 (1950). FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). SCHULMAN, J., JR., AND FALKENHEIY, M., Nucleon&x 3, No. 13 (1948). BARNUM, C. P., JARDETZKY, C. D., AND HALBERG, F., Texas Repts. Biol. and Med. 16, 134 (1957). PORTER, K. R., J. Histochem. and Cytochem. 2, 346 (1964). PALADE, G. E., 1. Biophys. Biochem. Cytology 1, 59 (1965). SLAUTTERBACK, D. B., Exptl. Cell Research 6, 173 (1953). HOWATSON, A. F., AND HAM, A. W., Cancer Research 16, 62 (1955). PETEFLMANN, M. L., HAMILTON, M. G., AND MIZEN, N. A., Cancer Research 14, 360 (1954). JARDETZKY, C. D., AND BARNUM, C. P., to be published. JEENER, R., AND SZAFARZ, D., Arch. Biochem. 26, 54 (1950). JEENER. R., Biochim. et Biophys. Acta 8, 270 (1962). CHANTRENNE, H., Biochim. et Biophys. Acta 1, 437 (1947). GOLDSTEIN, L., AND PLAUT, W., Proc. Natl. Acad. Sci. U. S. 41, 874 (1955). AELSTRBY, L., EULER, H., AND HEVESY, G., Arkiv Kemi, Mineral. Geol. Ais, No. 9 (1944). STEVENS, C. E., DAOUST, R., AND LEBLOND, C. P., J. Biol. Chem. 202, 177 (1953).
7. FRESCO, J. 8. JARDETZKY,
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20.
21. 22. 23, 24.
OF BIOCHEMISTRY
AND
BIOPHYSICS
67,
350-365 (19%‘)
Metabolic Activity and Intracellular Distribution of Nucleic Acid Phosphorus in Regenerating Liver1 Christine D. Jardetzky2 and Cyrus P. Barnum From the Department
of Physiological Chemistry, Medical Minneapolis, Minnesota
School,
Received July 9, 1956 INTRODUCTION
Differences in the intracellular distribution and metabolic activity of ribonucleic acid phosphorus (RNAP) of various cell fractions from normal adult mouse liver and mammary carcinoma have recently become apparent (l-3). In the caseof the tumor tissue the specific activity data have been consistent with a metabolic scheme involving the different cell RNA’s, the deoxyribonucleic acid (DNA), and an unknown immediate precursor pool. Based on this metabolic scheme, on the calculated specific activity-time curve of the unknown precursor, and on the assumptions of the steady state and metabolic homogeneity of the various nucleic acid phosphorus pools, the fraction that is renewed per hour was estimated. This was found to be 118,596, and 150%/hr. for the cytoplasmic particulate and supernatant RNA’s, and nuclear RNA, respectively (2). Other values reported in the literature or calculated from half-lives, based on the loss of radioactivity from the particular nucleic acid and on the assumption that the specific activity of the immediate precursor is zero, (l/(rate/total amount) = tl,Jln 2) are 1.72 for total rat liver RNA (4), 0.52 for regenerating rat liver total RNA purines (5), 0.31 for total RNA purines in liver from tumor-bearing rats (6), and 5.9 and 2.7%/hr., respectively, for nuclear RNA and cytoplasmic RNA adenine from adult mouseliver (7). The marked differences in the fraction of RNA that is renewed per hour may be partly 1 These investigations were supported by a grant-in-aid from the American Cancer Society. * Present address: Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena 4, California. 350
NUCLEIC
ACID PHOSPHORUS IN LIVER
351
due to the additional assumption in Refs. (5-7) that the specific activity of the immediate precursor was zero at the time during which the loss of radioactivity from the particular nucleic acid fraction occurred. For the purpose of comparing the nucleic acid metabolism of normally growing tissue to that of either normal adult liver or tumor tissue, regenerating liver was chosen for study. The results from a study of distribution of RNA in different cell fractions obtained by differential centrifugation 60 hr. after partial hepatectomy and those from a shortterm Ps2incorporation into the nucleic acids are reported in this communication. A mathematical analysis of the data in an attempt to determine possible precursor-product relationships and the calculation of minimum values for the per cent renewal of the various RNA’s are also included. Such limiting values are felt to be more realistic than those obtained on the assumption that the immediate precursor has zero specific activity, but at the same time are not subject to the hazard of trying to approximate the specific activity-time curve of an unknown precursor. EXPERIMENTAL Male ZBC mice3 between 1.5 and 2.5 months of age were partially hepatectomized as previously described (8). They were housed in groups of 5-10 in wooden boxes and given Purina Fox Chow ad libitum and 10% dextrose for drinking until the time of sacrifice. The animals were operated on always between 9 and 11 p.m. and were sacrificed between 9 a.m. and 2 p.m., 0.5-6 hr. after the injection of P3*, and 58-64 hr. after partial hepatectomy. The P3* solution received from the Oak Ridge National Laboratory was diluted with 0.857o NaCl, and an amount of 2 microcuries bc.)/O.Ol ml./g. body weight was administered intraperitoneally. Pools of livers from 3 to 4 mice were homogenized in alkalinized saline in a Potter-Elvehjem homogenizer. The final volume of the homogenate was 15 ml. An aliquot was taken for the determination of the inorganic phosphorus (IP) specific activity. The rest of the homogenate was then fractionated by differential centrifugation into the following fractions: nuclear (N) at 1400 X g for 5 min. (or at 23,000 X g for 5 min. when the mitochondrial fraction was not saved), mitochondrial (L) at 23,000 X g for 5 min., microsomal (M) at 23,000 X g for 1.5 hr., ultramicrosomal (U) at 100,000 X g for 1 hr., and supernatant (8).
The N pellet was further purified by repeated washings, 12-16, with cold 24/, citric acid followed by two quick washings with cold alkalinized saline (9). The 3 We are indebted experiments.
to Dr. J. J. Bittner
for supplying
the mice used in these
352
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
phospholipides were then extracted with 95% ethanol followed by treatment with hot Bloor’s reagent and an ethanol wash. These extracts were combined and evaporated to dryness, and the phospholipides were picked up in petroleum ether. The lipide-free nuclei were treated with cold 0.1 M bicarbonate-carbonate buffer, pH 10, which removed about 50% of the total RNA (9). This fraction of N-RNA is labeled N-RNA1 . It was further purified from traces of acid-soluble phosphorus (ASP) and DNAP by precipitation with an equal volume of cold 10% trichloroacetic acid (TCA), hydrolysis of RNA with 0.1 M NaOH, and a second precipitation with cold 10% TCA. The DNA of the N pellet, after N-RNA1 removal, was solubilized at 100°C. with 6% NaCl buffered at pH 10 and then precipitated with 2 vol. of ethanol. The DNA in this sediment was solubilized in hot 10% NaCl, reprecipitated with ethanol, and further purified by a Schmidt-Thannhauser extraction as previously described (9). In some cases the ethanoldyc NaCl-pH 10 supernatant containing the other half of the total N-RNA was saved, evaporated to dryness, and extracted with cold 5% TCA. This was called the N-RNA2 fraction.
Cytoplasm The tubes containing the M and U pellets were rinsed once with cold alkalinized saline. The neutral fat adhering to the sides of the tubes was removed with gauze, and the pellets were transferred quantitatively into centrifuge tubes using 10% NaCl. Solid NaCl was added to the S fraction to give a final concentration of 10%. The samples were then placed in a water bath at 100” to solubilize the RNA and denature the protein. The RNA in the 10% NaCl extracts was precipitated with 2 vol. of ethanol, re-extracted with hot 10% NaCI, and reprecipitated with ethanol, solubilized in cold distilled water, precipitated with cold 10% TCA, and finally hydrolyzed with 5% TCA as previously described (3). However, difficulty was encountered in precipitating, with cold TCA, the RNA that was dissolved in water. Therefore, in some experiments this step was substituted by washing the second alcoho1 precipitate with cold 5% TCA, hydrolyzing for 30 min. at 80-65” with 0.1 M NaOH, cooling, and precipitating the glycogen and traces of contaminating protein with cold ethanol-TCA mixture to give a final TCA and ethanol concentration of 5 and SS’%, respectively. The RNA purified according to the altered procedure had the same specific activity as that obtained from the older method except at the 0.5-hr. period with Paz when it was almost doubIed. The older method was used in all cases in purifying RNA for specific activity determinations at 0.5 hr. The phospholipides were extracted from the sediments of the different cytoplasmic fractions after the removal of RNA, and were purified as described under Nuclei. The protein-P fractions were obtained from the purified N pellet and M, U, and S fractions after the complete removal of lipides and nucleic acids (3). The radioactive samples and standards were prepared for counting and were counted with a Geiger-Miiller dipping tube as indicated previously (3). Phosphorus was determined by the method of Fiske and SubbaRow (10) on the same aliquotsused for counting. Specific activities are reported as counts/min./mmole P
NUCLEIC
ACID PHOSPHORUS
353
IN LIVER
as per cent of counts/min. injected/g. body weight (BCC).’ Relative specific activity refers to counts/min./~g. P as per cent of counts/min./~g. IP. For the quantitative estimation of nucleic acids, the constituents of the different cell fractions were precipitated and washed with cold 5% TCA, and hydrolyzed with hot TCA (1). An aliquot from whole homogenate was treated similarly. DNA and RNA were determined by the diphenylamine and orcinol-HCI reactions (1). The absorbancy of RNA solutions was corrected for the presence of DNA and glucose whenever necessary.6 These corrections never amounted to more than 5-10% of the absorbancy of the solutions. RESULTS
From Table I it can be seenthat about 43 % of the total cell RNA is associated with the microsome fraction. Approximately RNA can be accounted for. The incomplete recovery
70 % of the total is probably due to
loss of whole cells that escaped breakage in the process of homogenization and perhaps due to some hydrolysis of RNA into components during the period of sedimentation of the
acid-soluble various cell
fractions. The acid-soluble constituents of the various pellets are probably contaminated by those of the supernatant liquid trapped in the pellets. A large amount of the total cell phospholipide probably sediments at 1400 X g for 5 min. since only about 50% could be recovered from
the different
fractions.
It is interesting
to note that
the RNA
to
lipide-P ratio of the N pellet is approximately the same as that of the M pellet, which is about one-half that of the U and S fractions, and five times
that
of the L pellet.
The
amount
of IP/g.
tissue was found
to be
226 f 9 pg., and approximately 26 % of the total ASP. The specific activities of IP and the different nucleic acids are seenin Table II. Not shown in this table are the N-RNA* specific activities at 0.5, 1, and 2 hr. after Ps2injection; these are 1423 f 115, 1940 f 256, and 2834 f 724, respectively. Comparison of N-RNA1 and N-RNA, does not reveal any appreciable differences in their mean specific activity, although
each may be a mixture
of metabolically
distinct
RNA’a.
Also,
the specific activity of L-RNA at 0.5 and 1 hr. was almost identical with the mean specific activity of M-RNA at those times. The specific activity-time curves of the IP and the various nucleic acids are seen in Figs. 1 and 2. It is noted in Fig. 2 that the specific activity-time curve for DNA has a very small slope between 3 and 6 4 The use of the Biological Concentration mended by Schulman and Falkenheim (11). 6 C. P. Barnum, unpublished experiments.
Coefficient
(BCC) has been recom-
354
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
hr. This observation suggested that the rate of entrance of IP into the DNA precursors and/or the rate of DNA synthesis from these precursors might be decreased during the later interval of the kinetic study. Therefore, the 1-hr. incorporation of Pa2 into the DNA and other constituents was studied as a function of the time after the operation and the time of day when the animals were sacrificed. The results are given in Table III. It is seen that the relative specific activity of DNA is higher in the TABLE I Regenerating Liver Composition 60 Hours after Partial Hepatectomy Average values are based on 4-10 duplicate analyses. For each analysis pools of livers from 3 to 4 mice were used. RNA and DNA were determined with the orcinol-HCl and diphenylamine reactions. Micrograms/g. Cell fraction0
H
ASP
fresh liver RNA
f optimal Lipid&
estimateb RNA Lipid&
10,074 1,201 8.4 f406 f138 N 424 23 18.2 f63 f5 N= 590 32 L 23 390 2.9 135 f4 f50 f22 LC 40 668 231 M 15.2 62 4,340 286 f19 f541 f64 U 10 662 19 35.6 f3 f20 f4 S 720 919 30 31.0 f39 f34 f6 NC, Le, M, TJ, S 833 7,179 598 a H denotes homogenate; N, nuclei; L, mitochondria; M, microsomes; U, ultramicrosomes; S, supernatant; ASP, acid-soluble phosphorus. z(r - zp bf N-1. d c Constituents corrected for the loss of nuclei or mitochondria. The correction factor for nuclei was based on the quantitative estimation of DNA from H (2012 f 154agJg.) and that from N (1447 f 202pgJg.). An approximate correction factor for mitochondria was based on the RNA to lipide-P ratios of H (8.4), L (2.9), and of the combined citric acid washes of the N pellet (6.9). A preferential loss of mitochondria could decrease this ratio from 8.4 to 6.9, and the difference between the total amount of lipide-P (521 pg./g.) in the combined citric washes and 6.9/8.4 times this amount was attributed to the mitochondria. The RNA was 2.9 times the amount of iiplde-P. 879 3279
NUCLEIC
ACID
PHOSPHORUS
IN
TABLE II Nucleic Acid and IP Specijic Activities BCCb Hours
f
optimal
355
LIVER
in Regenerating
Livera
estimate
with Pa’
0.5
IP
DNA
15,155
19 f2 49 ZIG3
N-RNA1
M-RNA
U-RNA
S-RNA
19 51 fl,lSl fl f2 f12 1 11,841 32 73 144 fl,O90 f6 f21 f35 2 6,541 114 112 192 251 f859 f38 f13 *30 f22 3 4,679 167 230 310 408 *377 f24 zk.33 f32 f84 4 4,170 180 2,414 292 407 465 f128 f32 zk283 f22 f47 f26 6 3,068 200 2,041 389 481 545 f105 f21 f102 f28 f21 f47 a The mice were sacrificed 58-64 hr. postoperatively from 9 a.m. to 2 p.m. at times indicated after P32 injection. Pools of 3-4 livers were used for each analysis, and a total of 4-10 analyses per time point. b BCC = counts/min./mmole P as per cent of counts/min. administered/g. body weight.
0
I
1,120 zk67 2,075 f275 2,566 f228 2,583 f242
2 3 Time anhours4
6
5
6
FIG. 1. Specific activity-time curve for inorganic phosphorus (IP), nuclear (N) RNA, supematant (S) RNA, and DNA of mouse liver 58-64 hr. after partial hepatectomy. Vertical lines in this and each of the subsequent figures indicate f one standard error of the means. Biological Concentration Coefficient (BCC) refers to counts/min./mmole P as per cent of counts/min. injected/g. body weight.
356
CHRISTINE
D.
600
JARDETZKY
,
AND
CYRUS
P. BARNUM
t
I
500 !&!400 Cx
-
3 4 5 6 Time in hours FIG. 2. Specific activity-time curves for three cytoplasmic RNA’s, supernatant (S), ultramicrosomal (U), and microsomal (M) RNA and DNA of mouse liver 58-64 hr. after partial hepatectomy. 0
I
2
than in the early afternoon, although the difference was not found to be statistically significant (P = 0.1). More recent results on the incorporation of Pa2into the DNA of young ad l&turn fed mice show a threefold decrease in the 2-hr. relative specific activity of DNA at 8:30 p.m. compared with that at 4:30 a.m. (12). These results on immature growing livers indicate that between 8 a.m.
morning
One-Hour Time of sacri6ce 1 hr. with P*’
TABLE III Relative Specific Activity (RSA)= of Regenerating Liver Constituents at Different Times of Day RSA -I optimal estimate chntsjmhL/fJg. z;,B’
- ASPC
N-RNA1
IP
DNA
0.48 71 16.4 9:30 a.m., 69 hr. after 8 12.9 ~2.7 zkO.11 partial hepstectomy 70 15.3d 0.35 1:30 p.m., 65 hr. after 8 f3.2 ho.5 10.06 partial hepatectomy a RSA = counts/min./pg. P as per cent of counts/min./fig. * Pools of two livers were used for each analysis. = This is only the organic fraction of the ASP. d Average of three analyses.
2,350 1117 2,224 f213 IP.
P
NUCLEIC
ACID
PHOSPHORUS
IN
LIVER
357
and 2 p.m. one would expect about the decrease in Pa2 incorporation into DNA that is noted in Table III for the regenerating liver. In Tables IV and V the specific activities of the various phospholipides and protein-P fractions are seen. Their specific activities are of the same order of magnitude as those of N-RNA. It may be noted also that the nuclear lipide-P and protein-P fractions have a lower specific activity at all times than that of any of the cytoplasmic fractions. DISCUSSION
The distribution of nucleic acids in regenerating liver 60 hr. after partial hepatectomy is more like that of normal adult liver than mammary carcinoma (1, 2). Most of the RNA is present in the microsome fraction in the adult and 60-hr. regenerating liver, while in tumor a relatively high percentage of the total cell RNA (24) is found in the ultramicrosome fraction, and it is indistinguishable in its specific activity from the RNA of microsomes. The microsome fraction is thought to be composed of RNA-rich particles associated with cytoplasmic membrane fragments (13-15). It appears then, that there is a greater number of such particles freely dispersed in tumor than in adult or in 60-hr. regenerating liver cells, and that the RNA from the free cytoplasmic particles in tumor is in rapid equilibrium with that of the particles attached to the endoplasmic reticulum, because of their similar specific activities. Howatson and Ham (16) predicted from electron microscope studies that a smaller percentage of the total cell RNA would be found in the microsomes from liver tumor than from normal liver cells. Their prediction was based on the observation that the cytoplasm of liver tumors had fewer reticular structures and a greater number of freely dispersed particles than that of normal liver. Ultracentrifugal and electrophoretic analyses carried out by Petermann et al. (17) on cytoplasmic supernatants after removal of nuclei and mitochondria have shown a decrease in the total amount of microsomes and in a heavy “B” component, along with an elevation in the amounts of the lighter “C” and “E” components 2 days after partial hepatectomy in rats. The amounts of the latter components were back to normal levels 3 days after the operation. The shifts in the amounts of RNA associated with the various cell fractions noted under different physiological and pathological conditions suggest that certain metabolic precursor-product * In certain instances in the literature the term “microsome” encompasses both the ultramicrosomal and the microsomal fractions described here.
358
CHRISTINE
D. JARDETZKY TABLE
Specific
Activity
of Phospholipide of Regenerating
AND
N
M
0.5
824
963 f147 1,984 f373 2,759 +471 2,904 f243 3,122 1138 2,875 f355
2 3
2,184c 1200 2,604
4 6
2,465 f87
P. BARNUM
IV from Different Cell Fractions Liver0 BCC f optimal estimate
Hours with Pa*
1
CYRUS
U
s
1,031
1,003
1,897* f12
1,753* *3 2,427
2,639 f202 2,957
2,436 f201 2,855
2,856* *51
2,816* 4x54
a See Table II. * Average of two analyses. c Average of three analyses. TABLE Specific
Activity
Hours with Paz 0.5 1 2 3 4 6
V
of Protein-P from Different Cell Fractions of Regenerating Livers BCC * optimal estimate N 1,315 ~328 1,611 f243 1,150 f333 906 1,727 *293 1,226” f598
M 1,892 f216 2,386 f224 2,438 zt348 2,479 2,941 *350 2,553” f131
S 2,773* f322 3,44O* f300 2,654 3~664 2,701 2,454 f102 2,166” f154
Q See Table II. * Average of two analyses. c Average of three analyses.
relationships may be operating and should be the subject for further study. The rationale for carrying out the kinetic study between 58 and 64 hr. after partial hepatectomy was that the growth rate of the liver at
NUCLEIC
ACID
PHOSPHORUS
IN
359
LIVER
that time appeared to be less variable than at earlier times after the operation. This was based on the values of the 1-hr. relative specific activity of DNA which showed a peak at 36 hr. but remained fairly constant between 54 and 72 hr. postoperatively (8). Therefore, it seemed reasonable to assume that during that time (a) the various liver constituents were synthesized at constant rates, and (b) the net increase in the total amount of these substances during the experimental period was very small. These two assumptions imply that the metabolic system is very close to the steady state. In a separate communication (18), an equation of first order in specific activities is derived from which the rate of appearance of a polymer from a homogeneously labeled precursor may be calculated. It is also shown that for any quantitative mathematical treatment of the results, the following assumptions must be valid: 1) conditions (a) and (b) above, and Z?) absence of compartments which form a barrier for the complete and instantaneous mixing of a specific nucleic acid pool, for example, the N-RPr’A pool. Cell membranes in the liver do not allow mixing of the RN-4 of one cell with that of another. Therefore, if the mathematical treatment is to be valid, it must be assumed that the reactions from precursor to a specific RNA are identical in all cells. With these assumptions in mind several schemes were tested for agreement with the experimental data, some of which are shown below. A F? N-RNA
F? S-RNB
B F? M-RNA
e
S-RNA Q U-RNA
G? M-RNA
--+
DNA t
S-RNA e
X F’t U-RNA
M M-RNA I
--+
T G X F), N-RNA n S-RNA F1 U-RNA
*
M-RNA
SchemesA and C are patterned after Jeener’s earlier and later suggestions according to which N-RNA is the immediate precursor of S-RNA which in turn is the immediate precursor of particulate RNA (19) on one hand, and, on the other, the particulate and nonparticulate RNA’s are formed independently of one another (20). Scheme B depicts Chantrenne’s hypothesis that larger cytoplasmic particles are formed from smaller ones (21), and schemeD is almost identical with that suggested by Barnum et al. (2) for the nucleic acids from mammary carcinoma.
360
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
None of these schemes (nor several variations of them) were found to satisfy the data from regenerating liver. The only relationship that was in reasonable agreement with the data is the following one: F? IP 4
The specific-activity are
N-RNA
4
M-RNA
equations applying to this system in the steady state dM = z (N - M) dt
and
dN = 2 (I - N) + 2 dt Equations
(1) and (2) in the integrated
N = z(l
(M - N) form are
- Y) + vf (/.l - v)
where capital letters represent specific activities, Greek letters represent the areas under the specific activity-time curves estimated with the use of Simpson’s rule (2), the V’S represent rates, and n and m the amounts of N-RNA and M-RNA/g. fresh liver. The values of the constant vz/m obtained from Eq. (3) at 0.5, 1, 2, 3, 4, and 6 hr. after Pa2 injection are 0.0322, 0.0295, 0.0344, 0.0378, 0.0366, and 0.0334, respectively, with a mean value of 0.034. According to this scheme the microsomal RNA is formed from nuclear RNA at the rate of 3.4% per hour. In Fig. 3, which is a graphical solution of Eq. (4), it is seen that a fairly good straight line goes through the points. The constants vi/n and (Q/n are equal to 0.24 and 0.34, respectively, and are obtained from the intercept and the slope of the line described by Eq. (4). These results imply that the nuclear RNA is formed from IP and is diluted by M-RNA at the rates of 24 and 34 %/l-u-., respectively. Since the fractions v2/m and (ve2)/n were evaluated independently from each other from Eqs. (3) and (4), and if it is assumed that the ratio v~/(v-2) is close to
NUCLEIC
ACID
,
0
PHOSPHORUS
I
IO FIG. 3. Graphical
1
IN
361
LIVER
4
20
I
30
g! solution of Eq. (4).
unity, the ratio of the amounts of M- and N-RNA, m/n, must be at least equal to 10. From Table I it is seen that m/n = 7.4, which is in pretty good agreement with the value predicted from the metabolic scheme. However, these quantitative metabolic relationships must be accepted with reservation because of the assumptions implied in the mathematical analysis. In partial agreement with this metabolic scheme is the experimental evidence presented by Goldstein and Plaut (22) that the nuclear RNA is the precursor of cytoplasmic RNA but that this is an irreversible processin ameba cells. In an attempt to avoid making the assumption of homogeneity of a particular nucleic acid fraction and guessingat the immediate precursor, the specific activity-time curve of the liver inorganic phosphorus was used to obtain minimum values for the fraction that appears per hour, as shown in the previous communication (18). These minimum values for the various RNA’s are underlined in Table VI. It is interesting to note that a minimum value for the renewal rate of N-RNAP is 25 %/hr. This value is lower than that obtained from the metabolic schemeaccording to which nuclear RNA derives its phosphorus from the inorganic phosphorus and the microsomal RNA with the over-all rate of 58 %/hr., but it is higher than most values reported in the literature for either
362
CHRISTINE
D.
JARDETZKY
AND
CYRUS
P.
BARNUM
TABLE VI Approzimate Fractional Rates of Appearance of Various Ribonucleic Acid Phosphorus Fractionsa Fractional Time
with
Pap
N-RNA
rate/k
S-RNA
X 100 U-RNA
M-RNA
1.16 0.42 0.13 19.5 1.20 0.60 0.27 15.1 1.39 0.95 0.57 13.0 1.54 1.22 0.86 11.1 1.67 1.32 0.99 3.4 1.54 1.36 1.09 = Derived by substituting the area under the true immediate precursor specific activity-time curve by that of the IP of the liver (18). Minimum values for the fraction of nucleic acid phosphorus appearing per hour are the italicized maximum values. The assumptions of an essentially homogeneous IP pool in the liver and the steady state within the 6-hr. interval 53-64 hr. after partial hepatectomy are implied. 0%
24.7
1 2 3 4 6
total or nuclear RNA (4-7). For the cytoplasmic RNA’s the minimum values range from 1 to 2%/hr. The fact that U-RNA has a specific activity-time curve intermediate between those of M and S-RNA suggestedthe possibility that it may be a mixture of particles containing S and M RNA. In order to determine how much S and M RNA would give a mixture whose specific activitytime curve was similar to that found experimentally for U-RNA, the following equation was set up UU = u,S + u,,,M
(5)
where u is the amount of U-RNA/g. liver, us and u, are the amounts of U-RNA from the S and M RNA pools, respectively, and the capital letters are the observed specific activities of the RNA’s. Since u = us + U Vlk, Eq. (5) can be reduced to the following one with only ua as an unknown. lb=
u(V - M) S-M
(6)
From the amount of U-RNA (u = 662 pg./g. liver, Table I) and Eq. (6), us was calculated and was found to have the following values at 0.5, 1, 2, 3,4, and 6 hr.: 194, 242, 381, 299,442, and 389 with an average of 325 pg./g. Then, by difference, the average u,,, is 337 pg./g. Using the
NUCLEIC
ACID
PHOSPHORUS
TABLE
IN
VII
Observed and Calculated SpeciJic Activities Hours with P32 Observed BC0 Calculated
BCC”
= From Table II. b Obtained from Eq. (5) mixture of M- and S-RNA.
0.5 19 f2 28
1 73 f21 87
on the
basis
363
LIVER
2 192 zt30 180 that
U-RKA
(BCC) of U-RNA 3 310 f32 317
4 407 f47 377
is approximately
6 481 f21 466 a 50:50
average values of us and u, , the specific activity of U-RNA was calculated from Eq. (5). The calculated and observed values are given in Table VII. It is seenthat with the exception of the 0.5-hr. point, all the rest of the points fall within the range of f one optimal estimate of the mean. More experimental work, however, is necessary before one can conclude that the U fraction is composed of particles the RNA of which is a 50:50 mixture of S- and M-RNA. It is pertinent to mention that contamination of U from S-RNA due to the trapping of a small volume of supernatant fluid in the U pellet does not amount to more than 3 % of the total U-RNA. With regard to the DNA, it is seen from Table III that the 1-hr. relative specific activity may be lower in the early afternoon than in the morning. This is in agreement with recent results that establish a marked daily variation in the 2-hr. relative specific activity of the DNA and mitosis in the immature growing liver (12). Such a daily variation also affects the mitotic index and P32incorporation into the DNA of regenerating mouse liver (12). The morning-afternoon changes in the l-hr. relative specific activity of DNA (Table III), as well as the flatness in the DNA specific activity-time curve between 3 and 6 hr. after Ps2injection (Fig. 2), imply that the rate of DNA formation from its immediate precursor may not be constant during the 6-hr. interval of the kinetic study, since there is no change in the 1-hr. specific activity of either the IP or the N-RNAP. For this reason, it did not appear justifiable to assume the steady state for the rates of reactions leading to DNA synthesis, and metabolic schemesinvolving the DNA were not tested. The hypothesis, proposed originally by Ahlstrijm et al. (23), and later by Barnum et al. (2) and Stevens et al. (24), that the DNAP of growing cells may be metabolically unstable, remains to be tested in systems in which DNA synthesis proceeds at a constant rate and the specific activity-time curve of the immediate precursor is known. It should be
364
CHRISTINE
D. JARDETZKY
AND CYRUS P. BARNUM
mentioned that, in contrast to the decreasing trend in the 2-hr. relative specific activity of DNA between 8:30 a.m. and 4;30 p.m., the various RNA’s during this time exhibit fairly constant relative specific activities in the immature growing liver (12) Y ACKNOWLEDGMENTS The authors gratefully acknowledge the skillful assistance of Miss Marlys J,. Hawkinson and Mrs. Margaret M. Scheller in the many chemical and radioactive
analysesreported. SUMMARY
The specific activity-time curves for the various liver nucleic acids were determined within a 6-hr. period from 58 to 64 hr. after partial hepatectomy in mice using Paz as the tracer. The nuclear, supernatant, ultramicrosomal, and microsomal RNA’s are listed in the order of decreasing specific activities. The protein and phospholipide phosphorus specific activities increased rapidly during the first hour, and all, with the exception of those from nuclei that were lower, were almost equal to the IP specific activity 6 hr. after Pa2injection. A study of the quantitative distribution of RNA revealed that about 40% of the cell RNA in the 60-hr. regenerating liver is associated with the microsome fraction. Only about 5-10% could be accounted for by each of the RNA’s of the other fractions. Assuming the condition of the steady state and metabolic homogeneity of the various nucleic acid phosphorus pools, equations representing different metabolic schemes were tested. It was shown that the data were in reasonable agreement with a metabolic scheme according to which M-RNA is formed from N-RNA at the rate of 3.4%/hr., and N-RNA appears at the rate of 24 and 34%/hr. from IP and M-RNA, respectively. Limiting fractional rates for the appearance of the various RNA’s were calculated using the liver inorganic phosphorus specific activity-time curve in place of that of the immediate precursor; these were l-2 % and 25 %/hr. for cytoplasmic and nuclear RNA, respectively. This fractional renewal rate for N-RNA is much greater than most values reported in the literature and, even so, must be considered a limiting minimum value. It seemedunjustified to assumethe steady-state condition during the kinetic study for the rates of reactions leading to DNA on the basis of 1 Unpublished
results.
NUCLEIC
ACID
PHOSPHORUS
IN
LIVER
365
the shape of its specific activity-time curve and on the morning-afternoon differences in the 1-hr. relative specific activity. Therefore, metabolic reactions involving DNA were not tested. An equation was developed to test whether the U fraction is a mixture of particles containing M- and S-RNA. The calculated specific activities from approximately a 50:50 mixture of S- and M-RNA were in fairly good agreement with the observed values of U-RNA. REFERENCES 1. HUSEBY, 2. BARNUM,
R. A., AND BARNUM, C. P., Arch. Biochem. 26, 187 (1950). C. P., HUSEBY, R. A., AND VERMUND, H., Cancer Research
13, 880
(1963). 3. 4. 5. 6.
BARNUM, C. P., AND HUSEBY, R. A., Arch. Biochem. 29, 7 (1950). HAMMARSTEN, E., AND HEVESY, G., Acta Physiol. Stand. 11, 335 (1946). FURST, S. S., ROLL, P. M., AND BROWN, B. G., J. Biol. Chem. 183, 251 (1950). TYNER, E. P., HEIDELBERQER, C., AND LEPAGE, G. A., Cancer Research 12,
158 (1952). R., AND MARSHAK, A., J. Biol. Chem. 206, 585 (1963). C. D., BARNUM, C. P., AND VERMUND, H., J. Biol. Chem. 222, 421 (1956). BARNUM, C. P., NASH, C. W., JENNINGS, E., NY~AARD, O., AND VERMUND, H., Arch. Biochem. 26, 376 (1950). FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). SCHULMAN, J., JR., AND FALKENHEIY, M., Nucleon&x 3, No. 13 (1948). BARNUM, C. P., JARDETZKY, C. D., AND HALBERG, F., Texas Repts. Biol. and Med. 16, 134 (1957). PORTER, K. R., J. Histochem. and Cytochem. 2, 346 (1964). PALADE, G. E., 1. Biophys. Biochem. Cytology 1, 59 (1965). SLAUTTERBACK, D. B., Exptl. Cell Research 6, 173 (1953). HOWATSON, A. F., AND HAM, A. W., Cancer Research 16, 62 (1955). PETEFLMANN, M. L., HAMILTON, M. G., AND MIZEN, N. A., Cancer Research 14, 360 (1954). JARDETZKY, C. D., AND BARNUM, C. P., to be published. JEENER, R., AND SZAFARZ, D., Arch. Biochem. 26, 54 (1950). JEENER. R., Biochim. et Biophys. Acta 8, 270 (1962). CHANTRENNE, H., Biochim. et Biophys. Acta 1, 437 (1947). GOLDSTEIN, L., AND PLAUT, W., Proc. Natl. Acad. Sci. U. S. 41, 874 (1955). AELSTRBY, L., EULER, H., AND HEVESY, G., Arkiv Kemi, Mineral. Geol. Ais, No. 9 (1944). STEVENS, C. E., DAOUST, R., AND LEBLOND, C. P., J. Biol. Chem. 202, 177 (1953).
7. FRESCO, J. 8. JARDETZKY,
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20.
21. 22. 23, 24.