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Nuclear ribonucleic acid polymerase activity, rate of ribonucleic acid synthesis and hydrolysis during development. The role of glucocorticoids
NUCLEAR
RIBONUCLEIC
ACTIVITY,
RATE
THESIS MENT.
AND THE
OF
ACID
RIBONUCLEIC
HYDROLYSIS ROLE
POLYMERASE
OF
ACID
DURING
SYN-
DEVELOP-
GLUCOCORTICOIDS*
FABIO SERENI a n d OTTAVIO BARNABEI Department of Pediatrics of the University of Milano, Milano, Italy, and Institute of General Physiology of the University of Ferrara, Ferrara, Italy
INTRODUCTION
MOST of the work which was accomplished during the past decade on the peculiarities of protein synthesis during development was performed in early stages of embryonic life. The principal aim of these studies was to get a better understanding of the mechanisms by which differentiation does occur in a very primordial period of growth and development. They led to a great deal of important knowledge on both nucleic acid and protein synthesis, even if we are still very far from a comprehensive general theory on the biochemical mechanisms by which either genetic or environmental factors act regulating tissue differentiation processes, o) Comparatively very little is known on the rate of protein synthesis in much later periods of life, when, from a morphological point of view, the main cell types of various tissues are already present, but great functional differences may occur from one stage of development to a subsequent one. Undoubtedly, in mammals, the brisk passage from intrauterine to extrauterine life (i.e. birth) represents a very critical period of time, mainly because all of a series of new activities (some of them previously accomplished by placental or maternal tissues) must be performed very suddenly by neonatal tissues. In this respect, liver is to be specially considered for its key role in intermediary metabolism, and therefore in maintaining homeostasis. Previous studies in more detail by us and others have demonstrated that *The present work was supported by grants from the National Institute for Child Health and Human Development (HD 01895-01), from the Association for the Aid of Crippled Children and from Consiglio Nazionale delle Ricerche. 165
166
FABIO SERENI AND OTTAVIO BARNABEI
the activity of a number of enzyme systems varies deeply after birth in the liver of the rat and other laboratory animals. (2'3) It is reasonable to assume that this postnatal increase may be considered in some instances as a consequence of a sudden activation of the rate of synthesis of enzyme molecules. (4,5) This increase appears to be dependent, at least in the case of tyrosine-a-keto-glutarate transaminase, on newly formed RNA, since it can be prevented by injection of actinomycin D. (5) Starting from these preliminary considerations, we recently performed a series of investigations with the principal aim of evaluating the rate of nuclear ribonucleic acid synthesis and metabolism in the liver of the rat in the perinatal period3 6) The possible role of adrenal steroids in regulating RNA synthesis in the same period of life will be also discussed. The following series of experiments will be reported: evaluation of the rate of incorporation of pyrimidine precursors (6-14C-orotate and 2-14C-uridine) into nuclear R N A of the liver of the rat before and alter birth; the developmental curve of nuclear DNA-dependent RNA polymerase activity during the same period of life; the in vitro rate of hydrolysis of pulse labelled nuclear R N A of the liver of newborn and adult rats, at different periods after birth; the influence of adrenalectomy at birth on the rate of incorporation of pyrimidine precursors into nuclear RNA; the possibility of interfering with the rate of R N A synthesis and metabolism by injecting glucocorticoids into newborn and adult rats.
MATERIALS
AND METHODS
Albino rats, Wistar strain, were used in all experiments. The age of fetuses was calculated from the copulation day, with a maximum error of 12 hr. 6-14C-orotate and 2-14C-uridine were injected intraperitoneally into fetuses (through the uterine wall) and newborns to evaluate the rate of nuclear R N A synthesis. All the animals were killed by decapitation 20 min after the injection, if not differently specified. Separation of RNA from nuclear and cytoplasmic fractions was performed as previously reported (T) and radioactivity was evaluated by a thin-end window gas flow Geiger Mueller counter. Nuclear DNA-dependent R N A polymerase activity (nucleosidetriphosphate:RNA nucleotidyl-transferase E.C.2.7.7.6) was assayed essentially with the procedure described by Weiss. (s) Liver nuclei were isolated by the procedure of Sporn and Dingman. (a)
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
167
Hydrolysis of pulse labeled nuclear R N A was evaluated in vitro on isolated nuclei which were suspended in a medium as reported in Table 3. The reaction was stopped by adding an adequate amount of 10% TCA; after the usual washings, R N A was extracted twice at 100°C with 10% NaCI containing yeast R N A as carrier, precipitated with ethanol and assayed for radioactivity. D N A was estimated by the dipnenylamine procedure. (xo) R N A was assayed by the orcinol method. (1°) RESULTS
AND
DISCUSSION
Rate of Synthesis and Hydrolysis of Liver Nuclear R N A in Fetus and Newborn Rats The first series of data to be discussed in this regard concerns the rate of incorporation of pyrimidine precursors into nuclear RNA. They are summarized in Figs. 1 and 2. 9.000. .500 ODO0. .500
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From very low values of 6-14C-orotate incorporation in fetuses close to term and in newborn just after birth, the values rise very sharply starting 3 hr after birth to reach a maximum at about 72 hr of life. Similar results were obtained when pulse labelings of 20 min or 60 rain were employed, and the incorporation into cytoplasmic R N A followed a pattern similar to that into nuclear R N A (see Table 1).
168
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FIG. 2 Incorporation of radioactivity from 2-~4C-uridine into nuclear R N A of the liver of fetus, newborn and adult rats. (From Sereni e t al.(%
Radioactivity from 2-14C-uridine incorporation was also low before birth, but it rose sharply shortly after birth. Adult values are much lower when compared with those of growing animals, when labelled uridine was used instead of orotic acid. Although no adequate explanation can be 400 Z ~D
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NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
169
given, it s e e m s probable that the higher activity of uridine-phosphorylase in adult liver and its v e r y low activity at birth m a y be responsible for this result.(11) T h e question w h e t h e r or not the increased postnatal incorporation of radioactivity f r o m pyrimidine p r e c u r s o r s into nuclear R N A m a y b e interpreted as an index o f an activation of R N A synthesis is obviously o p e n to discussion. T h r e e main possibilities must in effect be taken into account: TABLE 1 Influence of Time on 6-~4C-OroticAcid Incorporation into Liver RNA of Newborn Rats 6J4C-orotic acid incorporation into RNA c.p.m./mg RNA Age
Exp. 1 (20 rain)
Exp. 2 (60 min)
nuclear fraction
cytoplasmic fraction
nuclear fraction
cytoplasmic fraction
3-4 hr
1624 1615
46 136
2896 2459
369 275
4 days
3324 3110
224 140
4596 5014
971 1090
Data of experiment 1 refer to incorporation values obtained from liver of animals sacrificed 20 rain after 6-~4C-orotic acid injection. Data of experiment 2 refer to values obtained from animals killed 60 rain after the injection. Each figure is the resultant of determinations performed on pool of two livers. the first one is that the high labeling of R N A is truly due to a faster rate o f nuclear R N A p o l y m e r a s e activity, the second one is that, in the brief period of time before and after birth, significant variations of the specific activity of the R N A p r e c u r s o r pool do effectively occur. Finally, there is also to be considered the possibility that the passage f r o m intrauterine to extrauterine life is followed b y a d e c r e a s e d rate of hydrolysis of newly formed R N A or o f s o m e precursors. Unfortunately, we do not have any direct m e a s u r e m e n t of the size of pyrimidine p r e c u r s o r pool o f the liver cells during the perinatal period o f life. It m a y be interesting to r e m e m b e r in this regard that Christensen and Clifford (12) h a v e s h o w n a sharp postnatal intensification o f hepatic accumulation o f amino acids in the guinea-pig, a finding which was interpreted as an index of a sudden increase o f permeability to organic molecules o f cell m e m b r a n e s after birth.
170
FAB10 SERENI AND OTTAVIO BARNABEI
Data from the literature concerning the rate of conversion of pyrimidine precursors into pyrimidine by the liver of rat fetuses at term are conflicting. Thus Bresnick et al. os'14) have found a reduced labelling of U M P in fetal liver after injection of 6-14C-orotate into the amniotic sac. On the other hand, very recently Kretchmer and Hurwitz o5) have recorded a very active rate of pyrimidine synthesis in slices of rat fetal liver incubated in vitro; such a function resulted, however, almost completely dependent on the availability in the incubating medium of phosphoribose pyrophosphate. It seems, therefore, probable that a limiting factor in pyrimidine synthesis may be acting in vivo as well. On the basis of the data by Van Rossum, o6) who found that in the last days of intrauterine life a depression in the respiratory rate takes place, it appears possible that such a limiting factor is a reduced disposal of ATP. If a reduced synthesis of pyrimidine nucleotides occurs in the liver of rat fetus at term, the obvious consequence would be an impaired R N A formation. Our data on R N A polymerase activity of liver nuclei in the rat before and after birth support the hypothesis that birth brings along an activation of liver R N A synthesis. From Fig. 3, it may be noticed that the enzyme activity is low before birth and at birth, but that it doubles in the first 12 hr of extrauterine life. The R N A polymerase values remain, however, lower than those usually found in adults till the growing rats reach the age of 20-25 days. No differences were found, as far as magnesium dependency, histone, DNAse and actinomycin inhibitory influences are concerned, when newborn and adult liver R N A polymerase were compared (see Table 2). Finally, in order to rule out the possibility that a different rate of R N A hydrolysis at various ages could significantly reduce the significance of our data on R N A synthesis, a series of experiments was performed as reported in Table 3. It appears that the rate of hydrolysis of both nuclear and microsomal RNA was slightly faster in newborn than in adult rats. As far as these in vitro data are a rough index of the in vivo rate of R N A hydrolysis (which is debatable), they cannot contribute to explain the low R N A polymerase activity which was found in newborn rat liver. From an overall evaluation of our data, it seems therefore likely that in the liver of the rat a sharp postnatal increase in nuclear RNA synthesis occurs. The hypothesis that from this sudden postnatal activation of nuclear R N A synthesis an increased formation of some specific enzyme molecules occurs seems to us reasonable. Our preliminary results on the possibility of preventing in part the increased rate of incorporation of pyrimidine precursors into RNA by adrenalectomizing newborn rats at birth further support this hypothesis. Actually, that in the rat the liver of fetuses at term has a reduced capacity of forming RNA and that this
NUCLEAR
RIBONUCLEIC
ACID
POLYMERASE
ACTIVITY
171
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capacity increases soon after birth is indicated, also by a report by Geschwind and Li. tlr) T h e y found that R N A : D N A ratio did not vary appreciably from the 20th day of gestation to the first day of extrauterine life; at the 5th day of life it was somewhat higher than at birth and at the 40th day was very impressively increased. In addition, from the data of G e s c h w i n d and Li, it may be calculated that the total amount of R N A per liver did not vary during the last days of intrauterine and the first day of extrauterine life. More recently, an increase of the R N A : D N A ratio from the 4th to the 14th day of life has been described in the rat liver by Winick and Noble3 is) TABLE3 Hydrolysis of Rapidly Labeled RNA of Isolated Rat Liver Nuclei* (from Sereni et al.~8~)
Experiment
Age of Incubation animals timer (days) (rain)
1
1
2
7
0 15 45 0
Activity of RNA counts/min % of RNA hydrolyzed 568 152 60 1388
15 45
368 138
0 73 89.5 0
73.5 90
3
20
0 15 45
1125 301 102
0 73 91
4
Adult
0 15 45
1700 824 517
0 51.5 70
*Five t~C or orotate-6-14C (specific activity 30mC/mmole) was injected intraperitoneally 20 min before sacrifice. Nuclei were then isolated as described in the text. tlncubation medium contained in a final vol. of 1.6 ml:0.4 ml of suspension of nuclei. Mg++0.4 ~moles: Tris-HCI buffer pH 8.1. 100/zmoles: phosphate buffer pH 8.1, 100 p.moles.
Role o f Adrenal Cortex on Regulation o f Liver Nuclear R N A Synthesis in N e w b o r n Rats In previous experiments performed some years ago by one of us on the development o f liver tyrosine-~-keto-glutarate transaminase activity, it was demonstrated, beside its sharp postnatal increase, also the possibility
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
I73
tO prevent it by adrenalectomizing newborn rats at birth. O°) Very recently, R'fiih~i and Suihkonen (z°) have similarly shown, working on the postnatal development of the urea cycle enzymes, that the sharp increase of the arginine synthetase activity may be almost completely prevented not only by puromycin administration, but also by adrenalectomizing newborn rats at birth. Starting from these two premises, it was considered interesting to investigate the role of adrenal function on the postnatal activation of R N A synthesis we observed. The results we obtained are reported in Table 4. It may be noticed that if adrenalectomy is done shortly after birth and if a sufficient time elapses before the animals are killed, a definite inhibition of 6-14C-orotic acid incorporation into nuclear R N A can be demonstrated. However, when adrenalectomy was performed more than 24 hr after birth, when the rate of R N A synthesis has already increased, no significant differences between operated and intact animals were observed. It seems therefore that the adrenal function is an important factor in conditioning the sharp postnatal activation of the liver nuclear R N A synthesis. A series of experiments is at present in progress in our laboratory to investigate the possibility of influencing by adrenalectomy the postnatal increase of the R N A polymerase activity of rat liver. Much more complicated appears to be the problem of the influence of exogenous glucocorticoids on the liver R N A metabolism of young growing rats. Previous studies conducted by US (7"21'22) have demonstrated both in vivo and in the isolated and perfused adult liver that glucocorticoids influence TABLE 4 Influence of Adrenalectomy Performed Just After Birth on 6-14C-Orotic Acid Incorporation into Nuclear R N A of Rat Liver
Experiment
Age* (hr)
Time between adrenalectomy and sacrifice (hr)
1 2 3 4
15 20 26 72
14-15 15-16 24-25 69-70
Specific activity of nuclear RNA (c.p.m./mg RNA)
Controls 2699 1130 1654 4010
Operated 1181 783 589 2347
% variation in adrenalectomized animals when compared with controls
-56 -31 -64 -41
*At sacrifice of the animals. Each incorporation value was obtained from a minimum of two to a maximum of eight animals.
174
FABIO SERENI AND OTTAVIO BARNABEI
R N A m e t a b o l i s m b y m o r e than one way. M o r e specifically, it has b e e n p r o v e d that cortisone increases the rate o f synthesis of R N A (as was suggested b y an increase o f R N A p o l y m e r a s e activity) and also that it plays a definite role in stabilizing R N A b y decreasing its rate o f hydrolysis (both on the nuclear and the m i c r o s o m a l fraction). A s a probable consequence o f both these factors, a sharp increase in the rate o f pyrimidine p r e c u r s o r incorporation into nuclear R N A is usually o b s e r v e d in adult rat liver under the influence of glucocorticoids. Recently, K e n n e y eta/., (23"24) in an analysis o f the nature of R N A synthesized b y liver cells under the influence o f hydrocortisone, h a v e concluded that the steroid increases the nuclear synthesis o f b o t h ribosomal and transfer R N A . W h e n cortisone is injected to intact suckling rats, no influence was o b s e r v e d on R N A p o l y m e r a s e activity w h e n the animals were less than 13 d a y s old (Table 5). A n increased incorporation o f pyrimidine precursors into liver R N A was, on the contrary, o b s e r v e d after cortisone t r e a t m e n t in n e w b o r n rats 4 days old; this increase was noted both in nuclear and m i c r o s o m a l fraction, but m o r e m a r k e d l y in the latter one (Table 6). N e i t h e r effect was noticed in n e w b o r n rats less than 4 days old. T h e increased rate o f p r e c u r s o r incorporations into R N A in 4-day-old
TABLE5 Serial Determinations on the Influence of Glucocorticoids on DNA-dependent RNA Polymerase Activity of Isolated Nuclei from Newborn and Adult Rat Livers (from Barnabei et al. ~z~)) #/zmoles AMP-14Cincorporated into RNA/mg DNA Age (days)
3 6 6 7 8 13 13 15 22 Adult Adult Adult
Glucocorticoid
Cortisone Cortisone Cortisone Cortisol Cortisone Cortisone Cortisol Cortisone Cortisone Cortisone Cortisone Cortisone
Control
Glucocorticoid treated *
67 178 132 240 205 180 124 290 301 238 258 320
68 154 150 263 160 400 393 615 496 379 440 480
*5 mg/100 g was given intraperitoneally 4 hr before the experiment; controls received physiological saline.
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
175
rats, without any R N A polymerase activation, may possibly be interpreted as a consequence of a stabilization of R N A by glucocorticoids, which was indeed recorded at that age. te2) TABLE 6 Effect Of Cortisone on 6-~*C-Orotate Incorporation into Nuclear and Microsomal R N A o f Newborn and Adult Rat Liver* (from Ottolenghi and Bamahei ~n)) Specific activity of R N A (c.p.mdmg) Age
Nuclear fraction Microsomad fraction Cortisone
4 hr 36 hr 4 days Adult
1400 3370 3500 4500
1600 2610 4300 9250
Cortisone -124 335 292
-150 663 708
*Each value is the average of two different experiments. For each experhnent five to eight newborn and two adult rats were used. The animals were in each instance divided into two groups: the first one received cortisone intraperitoneally 5 m g l l 0 0 g, the second one physiological saline. 5 t~C of 6-t~C-orotate were given to each animal intraperitoneally. Aninuds were killed 4 hr after the iniection of cortisone and 20 rnin after the injection oforotate.
CONCLUSIONS
From an overall evaluation of the data we reported, it seems to us that the following su~ciently proved conclusions may be drawn. The first one is represented by the demonstration of the crucial role exerted by birth on activating R N A synthesis in the liver of the rat. This may l~-obably be c,ons~dered the first step leading shortly afterwards to an increased synthesis of a selected number of protein enzyme molecules, whose activity rises as soon as the extrauterine life starts. Adrenal function plays a very important role in the postnatal increase of liver R N A synthesis, as it is also essential in the postnatal development of a certain number of enzyme activities. This is demonstrated by the decreased rate of incorporation of pyrimidine precursors into nuclear R N A in newborn rats adrenalectomized shortly after birth. The administration of hydrocortisone to newborn rats shortly after birth does not influence appreciably the rate of liver R N A synthesis. S t a ~ n g from the
176
FABIO SERENI AND OTTAVIO BARNABEI
4th d a y o f life it is suggested that glucocorticoids interfere with R N A m e t a b o l i s m mainly stabilizing newly formed R N A molecules.
SUMMARY In the rat liver birth is followed b y m a r k e d changes in R N A metabolism. T h e rate of incorporation o f pyrimidine precursors into nuclear R N A and the activity o f D N A - d e p e n d e n t R N A p o l y m e r a s e o f isolated nuclei increase sharply. T h e s e data, together with previous reports f r o m the literature, suggest the hypothesis that during the neonatal period a sharp activation o f liver R N A synthesis occurs. O n the other hand, the rate o f in vitro hydrolysis of endogenous R N A b y liver nuclei was found to be s o m e w h a t higher in n e w b o r n than in adult rats. It s e e m s that adrenal function plays a role in the postnatal activation o f R N A synthesis o f n e w b o r n rat liver. This is suggested b y a lower specific activity of nuclear R N A o f n e w b o r n rats adrenalectomized at birth than in controls. N e w b o r n rat liver b e h a v e s in a peculiar w a y also as far as its r e s p o n s e to glucocorticoids is concerned. Differently f r o m what is usually o b s e r v e d in adult animals, after hydrocortisone injection no increase o f pyrimidine p r e c u r s o r incorporation into nuclear R N A is o b s e r v e d in animals less than 4 days old and the D N A - d e p e n d e n t R N A p o l y m e r a s e activity does not change before the 13th d a y o f life.
REFERENCES 1. J. BRACHET,Nucleic acid in development. Symposium on specificity of cell differentiation and interaction, J. Cell. Comp. Physiol. 60, suppl. 1, 1-18 (1962). 2. N. KRETCHMER,R. GREE•BERG and F. SERENI, Biochemical basis of immaturity, Ann. Rev. Med. 14, 407--426 (1963). 3. F. SERENI and N. PRINOm, The development of enzyme systems, Pediatric Clinics of NorthAmerica 12, 515-534 (1965). 4. F. T. Igmr~NEY,Mechanism of hormonal control of rat liver tyrosine transaminase, Advances in Enzyme Regulation 1, 137-150 (1963). 5. O. (~REENGARD,The role of coenzymes, cortisone and RNA in the control of fiver enzyme levels,Advances in Enzyme Regulation 1, 61-76 (1963). 6. F. SERENI, L. PICENISEREI~I,V. TOMASl and O. BARNABEI, Synthesis of ribonucleic acid and nuclear RNA polymerase activity of rat liver during the neonatal period. In preparation. 7. O. BARNABEIand F. SERENI,Cortisol induced increase of tyrosine-alpha-ketoglutarate transaminase in isolated rat liver. Biochim. Biophys. Acta 91,239-247 (1964). 8. S. B. WEiss, Enzymatic incorporation of ribonucleoside triphosphate into the interpolynucleotide linkages of ribonucleic acid, Proc. Natl. Acad. Sci. 46, 1020-1030 (1960). 9. M. B. SPORN and W. DINGMAN,The fractionation and characterization of nuclear ribonucleic acid from rat liver, Biochim. Biophys. Acta 68, 387-400 (1963).
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
177
10. W. C. SCHNEIDER, Determination of nucleic acids in tissues by pentose analysis, pp. 680-684 in Methods in Enzymology 3 (S. P. COLOWICKand N. O. KXPLAN, eds.), Academic Press, New York (1957). 11. L. STEVENSand L. A. STOCKEN, Studies on enzymes involved in nucleic acid metabolism in foetal young and regenerating rat liver, Biochem. J. 87, 12-15 (1963). 12. H. N. CHR[STENSEN and J. B. CLIFFORD, Early postnatal intensification of hepatic accumulation of amino acids, J. Biol. Chem. 238, 1743-1745 (1963). 13. E. BRESNICK,J. SAGE and K. LANCLOS, Ribonuclease activity in hepatic nuclei during development, Biochim. Biophys. Acta 114, 631-633 (1966). 14. E. BgESNICK, K. LANCLOS and E. GONZALES, The biosynthesis of ribonucleic acid in the fiver of the rat fetus, in vivo, Biochim. Biophys. Acta 108, 568-5?? (1965). 15. N. KRETCHMERand R. HURWITZ, Personal communication. 16. G. D. V. VAN ROSSUM, Respiration and glycolysis in liver slices prepared from rats of different foetal and post-natal ages, Biochim. Biophys. Acta 74, 15-23 (1963). 17. I. GESCHWIND and C. H. LI, The nucleic acid content of fetal rat liver, J. Biol. Chem. 170, 467-471 (1949). 18. M. WtNICK and A. NOBLE, Quantitative changes in D N A , R N A and protein during prenatal and postnatal growth in the rat, Developmental Biology 12, 451-466 (1965). 19. F. SERENI, F. T. K ~ N E Y and N. KRETCHMER, Factors influencing the development of tyrosine-alpha-ketoglutarate transaminase activity in rat liver, J. Biol. Chem. 234, 609-612 (1959). 20. N. C. R. RX,HX and J. SUIHKO~EN. Development of enzymes for urea biosynthesis in rat and human liver. Paper presented at the 36th annual meeting of the Society for Pediatric Research, Atlantic City, April 29-30 (1966). 21. O. BARNABE1, B. ROMANO, G. DI BITONTO, V. TOMAS1 and F. SERENI, Factors influencing the glucocorticoid-induced increase of ribonucleic acid polymerase activity of rat fiver nuclei, Arch. Biochem. Biophys. 113, 478-486 (1966). 22. C. OTTOI.ENGH1 and O. BARNABEI, Glucocorticoid control of the hydrolysis of pulse labelled microsomal ribonucleic acid and its possible significance in the hormonal response of rat liver. In preparation. 23. W. D. WICKS, D. L. GREENMAN and F. T. KENNEY, Stimulation of ribonucleic acid synthesis by steroid hormones. I. Transfer ribonucleic acid, .l. Biol. Chem. 240, 44144419 (1965). 24. D. U GaEENMAN, W. D. WICgS and F. T. KEN~EV, Stimulation of ribonucleic acid synthesis by steroid hormones. II. High molecular weight components, J. Biol. Chem. 240, 4420-4426 (1965).
RIBONUCLEIC
ACTIVITY,
RATE
THESIS MENT.
AND THE
OF
ACID
RIBONUCLEIC
HYDROLYSIS ROLE
POLYMERASE
OF
ACID
DURING
SYN-
DEVELOP-
GLUCOCORTICOIDS*
FABIO SERENI a n d OTTAVIO BARNABEI Department of Pediatrics of the University of Milano, Milano, Italy, and Institute of General Physiology of the University of Ferrara, Ferrara, Italy
INTRODUCTION
MOST of the work which was accomplished during the past decade on the peculiarities of protein synthesis during development was performed in early stages of embryonic life. The principal aim of these studies was to get a better understanding of the mechanisms by which differentiation does occur in a very primordial period of growth and development. They led to a great deal of important knowledge on both nucleic acid and protein synthesis, even if we are still very far from a comprehensive general theory on the biochemical mechanisms by which either genetic or environmental factors act regulating tissue differentiation processes, o) Comparatively very little is known on the rate of protein synthesis in much later periods of life, when, from a morphological point of view, the main cell types of various tissues are already present, but great functional differences may occur from one stage of development to a subsequent one. Undoubtedly, in mammals, the brisk passage from intrauterine to extrauterine life (i.e. birth) represents a very critical period of time, mainly because all of a series of new activities (some of them previously accomplished by placental or maternal tissues) must be performed very suddenly by neonatal tissues. In this respect, liver is to be specially considered for its key role in intermediary metabolism, and therefore in maintaining homeostasis. Previous studies in more detail by us and others have demonstrated that *The present work was supported by grants from the National Institute for Child Health and Human Development (HD 01895-01), from the Association for the Aid of Crippled Children and from Consiglio Nazionale delle Ricerche. 165
166
FABIO SERENI AND OTTAVIO BARNABEI
the activity of a number of enzyme systems varies deeply after birth in the liver of the rat and other laboratory animals. (2'3) It is reasonable to assume that this postnatal increase may be considered in some instances as a consequence of a sudden activation of the rate of synthesis of enzyme molecules. (4,5) This increase appears to be dependent, at least in the case of tyrosine-a-keto-glutarate transaminase, on newly formed RNA, since it can be prevented by injection of actinomycin D. (5) Starting from these preliminary considerations, we recently performed a series of investigations with the principal aim of evaluating the rate of nuclear ribonucleic acid synthesis and metabolism in the liver of the rat in the perinatal period3 6) The possible role of adrenal steroids in regulating RNA synthesis in the same period of life will be also discussed. The following series of experiments will be reported: evaluation of the rate of incorporation of pyrimidine precursors (6-14C-orotate and 2-14C-uridine) into nuclear R N A of the liver of the rat before and alter birth; the developmental curve of nuclear DNA-dependent RNA polymerase activity during the same period of life; the in vitro rate of hydrolysis of pulse labelled nuclear R N A of the liver of newborn and adult rats, at different periods after birth; the influence of adrenalectomy at birth on the rate of incorporation of pyrimidine precursors into nuclear RNA; the possibility of interfering with the rate of R N A synthesis and metabolism by injecting glucocorticoids into newborn and adult rats.
MATERIALS
AND METHODS
Albino rats, Wistar strain, were used in all experiments. The age of fetuses was calculated from the copulation day, with a maximum error of 12 hr. 6-14C-orotate and 2-14C-uridine were injected intraperitoneally into fetuses (through the uterine wall) and newborns to evaluate the rate of nuclear R N A synthesis. All the animals were killed by decapitation 20 min after the injection, if not differently specified. Separation of RNA from nuclear and cytoplasmic fractions was performed as previously reported (T) and radioactivity was evaluated by a thin-end window gas flow Geiger Mueller counter. Nuclear DNA-dependent R N A polymerase activity (nucleosidetriphosphate:RNA nucleotidyl-transferase E.C.2.7.7.6) was assayed essentially with the procedure described by Weiss. (s) Liver nuclei were isolated by the procedure of Sporn and Dingman. (a)
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
167
Hydrolysis of pulse labeled nuclear R N A was evaluated in vitro on isolated nuclei which were suspended in a medium as reported in Table 3. The reaction was stopped by adding an adequate amount of 10% TCA; after the usual washings, R N A was extracted twice at 100°C with 10% NaCI containing yeast R N A as carrier, precipitated with ethanol and assayed for radioactivity. D N A was estimated by the dipnenylamine procedure. (xo) R N A was assayed by the orcinol method. (1°) RESULTS
AND
DISCUSSION
Rate of Synthesis and Hydrolysis of Liver Nuclear R N A in Fetus and Newborn Rats The first series of data to be discussed in this regard concerns the rate of incorporation of pyrimidine precursors into nuclear RNA. They are summarized in Figs. 1 and 2. 9.000. .500 ODO0. .500
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From very low values of 6-14C-orotate incorporation in fetuses close to term and in newborn just after birth, the values rise very sharply starting 3 hr after birth to reach a maximum at about 72 hr of life. Similar results were obtained when pulse labelings of 20 min or 60 rain were employed, and the incorporation into cytoplasmic R N A followed a pattern similar to that into nuclear R N A (see Table 1).
168
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Radioactivity from 2-14C-uridine incorporation was also low before birth, but it rose sharply shortly after birth. Adult values are much lower when compared with those of growing animals, when labelled uridine was used instead of orotic acid. Although no adequate explanation can be 400 Z ~D
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NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
169
given, it s e e m s probable that the higher activity of uridine-phosphorylase in adult liver and its v e r y low activity at birth m a y be responsible for this result.(11) T h e question w h e t h e r or not the increased postnatal incorporation of radioactivity f r o m pyrimidine p r e c u r s o r s into nuclear R N A m a y b e interpreted as an index o f an activation of R N A synthesis is obviously o p e n to discussion. T h r e e main possibilities must in effect be taken into account: TABLE 1 Influence of Time on 6-~4C-OroticAcid Incorporation into Liver RNA of Newborn Rats 6J4C-orotic acid incorporation into RNA c.p.m./mg RNA Age
Exp. 1 (20 rain)
Exp. 2 (60 min)
nuclear fraction
cytoplasmic fraction
nuclear fraction
cytoplasmic fraction
3-4 hr
1624 1615
46 136
2896 2459
369 275
4 days
3324 3110
224 140
4596 5014
971 1090
Data of experiment 1 refer to incorporation values obtained from liver of animals sacrificed 20 rain after 6-~4C-orotic acid injection. Data of experiment 2 refer to values obtained from animals killed 60 rain after the injection. Each figure is the resultant of determinations performed on pool of two livers. the first one is that the high labeling of R N A is truly due to a faster rate o f nuclear R N A p o l y m e r a s e activity, the second one is that, in the brief period of time before and after birth, significant variations of the specific activity of the R N A p r e c u r s o r pool do effectively occur. Finally, there is also to be considered the possibility that the passage f r o m intrauterine to extrauterine life is followed b y a d e c r e a s e d rate of hydrolysis of newly formed R N A or o f s o m e precursors. Unfortunately, we do not have any direct m e a s u r e m e n t of the size of pyrimidine p r e c u r s o r pool o f the liver cells during the perinatal period o f life. It m a y be interesting to r e m e m b e r in this regard that Christensen and Clifford (12) h a v e s h o w n a sharp postnatal intensification o f hepatic accumulation o f amino acids in the guinea-pig, a finding which was interpreted as an index of a sudden increase o f permeability to organic molecules o f cell m e m b r a n e s after birth.
170
FAB10 SERENI AND OTTAVIO BARNABEI
Data from the literature concerning the rate of conversion of pyrimidine precursors into pyrimidine by the liver of rat fetuses at term are conflicting. Thus Bresnick et al. os'14) have found a reduced labelling of U M P in fetal liver after injection of 6-14C-orotate into the amniotic sac. On the other hand, very recently Kretchmer and Hurwitz o5) have recorded a very active rate of pyrimidine synthesis in slices of rat fetal liver incubated in vitro; such a function resulted, however, almost completely dependent on the availability in the incubating medium of phosphoribose pyrophosphate. It seems, therefore, probable that a limiting factor in pyrimidine synthesis may be acting in vivo as well. On the basis of the data by Van Rossum, o6) who found that in the last days of intrauterine life a depression in the respiratory rate takes place, it appears possible that such a limiting factor is a reduced disposal of ATP. If a reduced synthesis of pyrimidine nucleotides occurs in the liver of rat fetus at term, the obvious consequence would be an impaired R N A formation. Our data on R N A polymerase activity of liver nuclei in the rat before and after birth support the hypothesis that birth brings along an activation of liver R N A synthesis. From Fig. 3, it may be noticed that the enzyme activity is low before birth and at birth, but that it doubles in the first 12 hr of extrauterine life. The R N A polymerase values remain, however, lower than those usually found in adults till the growing rats reach the age of 20-25 days. No differences were found, as far as magnesium dependency, histone, DNAse and actinomycin inhibitory influences are concerned, when newborn and adult liver R N A polymerase were compared (see Table 2). Finally, in order to rule out the possibility that a different rate of R N A hydrolysis at various ages could significantly reduce the significance of our data on R N A synthesis, a series of experiments was performed as reported in Table 3. It appears that the rate of hydrolysis of both nuclear and microsomal RNA was slightly faster in newborn than in adult rats. As far as these in vitro data are a rough index of the in vivo rate of R N A hydrolysis (which is debatable), they cannot contribute to explain the low R N A polymerase activity which was found in newborn rat liver. From an overall evaluation of our data, it seems therefore likely that in the liver of the rat a sharp postnatal increase in nuclear RNA synthesis occurs. The hypothesis that from this sudden postnatal activation of nuclear R N A synthesis an increased formation of some specific enzyme molecules occurs seems to us reasonable. Our preliminary results on the possibility of preventing in part the increased rate of incorporation of pyrimidine precursors into RNA by adrenalectomizing newborn rats at birth further support this hypothesis. Actually, that in the rat the liver of fetuses at term has a reduced capacity of forming RNA and that this
NUCLEAR
RIBONUCLEIC
ACID
POLYMERASE
ACTIVITY
171
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capacity increases soon after birth is indicated, also by a report by Geschwind and Li. tlr) T h e y found that R N A : D N A ratio did not vary appreciably from the 20th day of gestation to the first day of extrauterine life; at the 5th day of life it was somewhat higher than at birth and at the 40th day was very impressively increased. In addition, from the data of G e s c h w i n d and Li, it may be calculated that the total amount of R N A per liver did not vary during the last days of intrauterine and the first day of extrauterine life. More recently, an increase of the R N A : D N A ratio from the 4th to the 14th day of life has been described in the rat liver by Winick and Noble3 is) TABLE3 Hydrolysis of Rapidly Labeled RNA of Isolated Rat Liver Nuclei* (from Sereni et al.~8~)
Experiment
Age of Incubation animals timer (days) (rain)
1
1
2
7
0 15 45 0
Activity of RNA counts/min % of RNA hydrolyzed 568 152 60 1388
15 45
368 138
0 73 89.5 0
73.5 90
3
20
0 15 45
1125 301 102
0 73 91
4
Adult
0 15 45
1700 824 517
0 51.5 70
*Five t~C or orotate-6-14C (specific activity 30mC/mmole) was injected intraperitoneally 20 min before sacrifice. Nuclei were then isolated as described in the text. tlncubation medium contained in a final vol. of 1.6 ml:0.4 ml of suspension of nuclei. Mg++0.4 ~moles: Tris-HCI buffer pH 8.1. 100/zmoles: phosphate buffer pH 8.1, 100 p.moles.
Role o f Adrenal Cortex on Regulation o f Liver Nuclear R N A Synthesis in N e w b o r n Rats In previous experiments performed some years ago by one of us on the development o f liver tyrosine-~-keto-glutarate transaminase activity, it was demonstrated, beside its sharp postnatal increase, also the possibility
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
I73
tO prevent it by adrenalectomizing newborn rats at birth. O°) Very recently, R'fiih~i and Suihkonen (z°) have similarly shown, working on the postnatal development of the urea cycle enzymes, that the sharp increase of the arginine synthetase activity may be almost completely prevented not only by puromycin administration, but also by adrenalectomizing newborn rats at birth. Starting from these two premises, it was considered interesting to investigate the role of adrenal function on the postnatal activation of R N A synthesis we observed. The results we obtained are reported in Table 4. It may be noticed that if adrenalectomy is done shortly after birth and if a sufficient time elapses before the animals are killed, a definite inhibition of 6-14C-orotic acid incorporation into nuclear R N A can be demonstrated. However, when adrenalectomy was performed more than 24 hr after birth, when the rate of R N A synthesis has already increased, no significant differences between operated and intact animals were observed. It seems therefore that the adrenal function is an important factor in conditioning the sharp postnatal activation of the liver nuclear R N A synthesis. A series of experiments is at present in progress in our laboratory to investigate the possibility of influencing by adrenalectomy the postnatal increase of the R N A polymerase activity of rat liver. Much more complicated appears to be the problem of the influence of exogenous glucocorticoids on the liver R N A metabolism of young growing rats. Previous studies conducted by US (7"21'22) have demonstrated both in vivo and in the isolated and perfused adult liver that glucocorticoids influence TABLE 4 Influence of Adrenalectomy Performed Just After Birth on 6-14C-Orotic Acid Incorporation into Nuclear R N A of Rat Liver
Experiment
Age* (hr)
Time between adrenalectomy and sacrifice (hr)
1 2 3 4
15 20 26 72
14-15 15-16 24-25 69-70
Specific activity of nuclear RNA (c.p.m./mg RNA)
Controls 2699 1130 1654 4010
Operated 1181 783 589 2347
% variation in adrenalectomized animals when compared with controls
-56 -31 -64 -41
*At sacrifice of the animals. Each incorporation value was obtained from a minimum of two to a maximum of eight animals.
174
FABIO SERENI AND OTTAVIO BARNABEI
R N A m e t a b o l i s m b y m o r e than one way. M o r e specifically, it has b e e n p r o v e d that cortisone increases the rate o f synthesis of R N A (as was suggested b y an increase o f R N A p o l y m e r a s e activity) and also that it plays a definite role in stabilizing R N A b y decreasing its rate o f hydrolysis (both on the nuclear and the m i c r o s o m a l fraction). A s a probable consequence o f both these factors, a sharp increase in the rate o f pyrimidine p r e c u r s o r incorporation into nuclear R N A is usually o b s e r v e d in adult rat liver under the influence of glucocorticoids. Recently, K e n n e y eta/., (23"24) in an analysis o f the nature of R N A synthesized b y liver cells under the influence o f hydrocortisone, h a v e concluded that the steroid increases the nuclear synthesis o f b o t h ribosomal and transfer R N A . W h e n cortisone is injected to intact suckling rats, no influence was o b s e r v e d on R N A p o l y m e r a s e activity w h e n the animals were less than 13 d a y s old (Table 5). A n increased incorporation o f pyrimidine precursors into liver R N A was, on the contrary, o b s e r v e d after cortisone t r e a t m e n t in n e w b o r n rats 4 days old; this increase was noted both in nuclear and m i c r o s o m a l fraction, but m o r e m a r k e d l y in the latter one (Table 6). N e i t h e r effect was noticed in n e w b o r n rats less than 4 days old. T h e increased rate o f p r e c u r s o r incorporations into R N A in 4-day-old
TABLE5 Serial Determinations on the Influence of Glucocorticoids on DNA-dependent RNA Polymerase Activity of Isolated Nuclei from Newborn and Adult Rat Livers (from Barnabei et al. ~z~)) #/zmoles AMP-14Cincorporated into RNA/mg DNA Age (days)
3 6 6 7 8 13 13 15 22 Adult Adult Adult
Glucocorticoid
Cortisone Cortisone Cortisone Cortisol Cortisone Cortisone Cortisol Cortisone Cortisone Cortisone Cortisone Cortisone
Control
Glucocorticoid treated *
67 178 132 240 205 180 124 290 301 238 258 320
68 154 150 263 160 400 393 615 496 379 440 480
*5 mg/100 g was given intraperitoneally 4 hr before the experiment; controls received physiological saline.
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
175
rats, without any R N A polymerase activation, may possibly be interpreted as a consequence of a stabilization of R N A by glucocorticoids, which was indeed recorded at that age. te2) TABLE 6 Effect Of Cortisone on 6-~*C-Orotate Incorporation into Nuclear and Microsomal R N A o f Newborn and Adult Rat Liver* (from Ottolenghi and Bamahei ~n)) Specific activity of R N A (c.p.mdmg) Age
Nuclear fraction Microsomad fraction Cortisone
4 hr 36 hr 4 days Adult
1400 3370 3500 4500
1600 2610 4300 9250
Cortisone -124 335 292
-150 663 708
*Each value is the average of two different experiments. For each experhnent five to eight newborn and two adult rats were used. The animals were in each instance divided into two groups: the first one received cortisone intraperitoneally 5 m g l l 0 0 g, the second one physiological saline. 5 t~C of 6-t~C-orotate were given to each animal intraperitoneally. Aninuds were killed 4 hr after the iniection of cortisone and 20 rnin after the injection oforotate.
CONCLUSIONS
From an overall evaluation of the data we reported, it seems to us that the following su~ciently proved conclusions may be drawn. The first one is represented by the demonstration of the crucial role exerted by birth on activating R N A synthesis in the liver of the rat. This may l~-obably be c,ons~dered the first step leading shortly afterwards to an increased synthesis of a selected number of protein enzyme molecules, whose activity rises as soon as the extrauterine life starts. Adrenal function plays a very important role in the postnatal increase of liver R N A synthesis, as it is also essential in the postnatal development of a certain number of enzyme activities. This is demonstrated by the decreased rate of incorporation of pyrimidine precursors into nuclear R N A in newborn rats adrenalectomized shortly after birth. The administration of hydrocortisone to newborn rats shortly after birth does not influence appreciably the rate of liver R N A synthesis. S t a ~ n g from the
176
FABIO SERENI AND OTTAVIO BARNABEI
4th d a y o f life it is suggested that glucocorticoids interfere with R N A m e t a b o l i s m mainly stabilizing newly formed R N A molecules.
SUMMARY In the rat liver birth is followed b y m a r k e d changes in R N A metabolism. T h e rate of incorporation o f pyrimidine precursors into nuclear R N A and the activity o f D N A - d e p e n d e n t R N A p o l y m e r a s e o f isolated nuclei increase sharply. T h e s e data, together with previous reports f r o m the literature, suggest the hypothesis that during the neonatal period a sharp activation o f liver R N A synthesis occurs. O n the other hand, the rate o f in vitro hydrolysis of endogenous R N A b y liver nuclei was found to be s o m e w h a t higher in n e w b o r n than in adult rats. It s e e m s that adrenal function plays a role in the postnatal activation o f R N A synthesis o f n e w b o r n rat liver. This is suggested b y a lower specific activity of nuclear R N A o f n e w b o r n rats adrenalectomized at birth than in controls. N e w b o r n rat liver b e h a v e s in a peculiar w a y also as far as its r e s p o n s e to glucocorticoids is concerned. Differently f r o m what is usually o b s e r v e d in adult animals, after hydrocortisone injection no increase o f pyrimidine p r e c u r s o r incorporation into nuclear R N A is o b s e r v e d in animals less than 4 days old and the D N A - d e p e n d e n t R N A p o l y m e r a s e activity does not change before the 13th d a y o f life.
REFERENCES 1. J. BRACHET,Nucleic acid in development. Symposium on specificity of cell differentiation and interaction, J. Cell. Comp. Physiol. 60, suppl. 1, 1-18 (1962). 2. N. KRETCHMER,R. GREE•BERG and F. SERENI, Biochemical basis of immaturity, Ann. Rev. Med. 14, 407--426 (1963). 3. F. SERENI and N. PRINOm, The development of enzyme systems, Pediatric Clinics of NorthAmerica 12, 515-534 (1965). 4. F. T. Igmr~NEY,Mechanism of hormonal control of rat liver tyrosine transaminase, Advances in Enzyme Regulation 1, 137-150 (1963). 5. O. (~REENGARD,The role of coenzymes, cortisone and RNA in the control of fiver enzyme levels,Advances in Enzyme Regulation 1, 61-76 (1963). 6. F. SERENI, L. PICENISEREI~I,V. TOMASl and O. BARNABEI, Synthesis of ribonucleic acid and nuclear RNA polymerase activity of rat liver during the neonatal period. In preparation. 7. O. BARNABEIand F. SERENI,Cortisol induced increase of tyrosine-alpha-ketoglutarate transaminase in isolated rat liver. Biochim. Biophys. Acta 91,239-247 (1964). 8. S. B. WEiss, Enzymatic incorporation of ribonucleoside triphosphate into the interpolynucleotide linkages of ribonucleic acid, Proc. Natl. Acad. Sci. 46, 1020-1030 (1960). 9. M. B. SPORN and W. DINGMAN,The fractionation and characterization of nuclear ribonucleic acid from rat liver, Biochim. Biophys. Acta 68, 387-400 (1963).
NUCLEAR RIBONUCLEIC ACID POLYMERASE ACTIVITY
177
10. W. C. SCHNEIDER, Determination of nucleic acids in tissues by pentose analysis, pp. 680-684 in Methods in Enzymology 3 (S. P. COLOWICKand N. O. KXPLAN, eds.), Academic Press, New York (1957). 11. L. STEVENSand L. A. STOCKEN, Studies on enzymes involved in nucleic acid metabolism in foetal young and regenerating rat liver, Biochem. J. 87, 12-15 (1963). 12. H. N. CHR[STENSEN and J. B. CLIFFORD, Early postnatal intensification of hepatic accumulation of amino acids, J. Biol. Chem. 238, 1743-1745 (1963). 13. E. BRESNICK,J. SAGE and K. LANCLOS, Ribonuclease activity in hepatic nuclei during development, Biochim. Biophys. Acta 114, 631-633 (1966). 14. E. BgESNICK, K. LANCLOS and E. GONZALES, The biosynthesis of ribonucleic acid in the fiver of the rat fetus, in vivo, Biochim. Biophys. Acta 108, 568-5?? (1965). 15. N. KRETCHMERand R. HURWITZ, Personal communication. 16. G. D. V. VAN ROSSUM, Respiration and glycolysis in liver slices prepared from rats of different foetal and post-natal ages, Biochim. Biophys. Acta 74, 15-23 (1963). 17. I. GESCHWIND and C. H. LI, The nucleic acid content of fetal rat liver, J. Biol. Chem. 170, 467-471 (1949). 18. M. WtNICK and A. NOBLE, Quantitative changes in D N A , R N A and protein during prenatal and postnatal growth in the rat, Developmental Biology 12, 451-466 (1965). 19. F. SERENI, F. T. K ~ N E Y and N. KRETCHMER, Factors influencing the development of tyrosine-alpha-ketoglutarate transaminase activity in rat liver, J. Biol. Chem. 234, 609-612 (1959). 20. N. C. R. RX,HX and J. SUIHKO~EN. Development of enzymes for urea biosynthesis in rat and human liver. Paper presented at the 36th annual meeting of the Society for Pediatric Research, Atlantic City, April 29-30 (1966). 21. O. BARNABE1, B. ROMANO, G. DI BITONTO, V. TOMAS1 and F. SERENI, Factors influencing the glucocorticoid-induced increase of ribonucleic acid polymerase activity of rat fiver nuclei, Arch. Biochem. Biophys. 113, 478-486 (1966). 22. C. OTTOI.ENGH1 and O. BARNABEI, Glucocorticoid control of the hydrolysis of pulse labelled microsomal ribonucleic acid and its possible significance in the hormonal response of rat liver. In preparation. 23. W. D. WICKS, D. L. GREENMAN and F. T. KENNEY, Stimulation of ribonucleic acid synthesis by steroid hormones. I. Transfer ribonucleic acid, .l. Biol. Chem. 240, 44144419 (1965). 24. D. U GaEENMAN, W. D. WICgS and F. T. KEN~EV, Stimulation of ribonucleic acid synthesis by steroid hormones. II. High molecular weight components, J. Biol. Chem. 240, 4420-4426 (1965).