nucleotide metabolism · nucleotide metabolism vi. the phosphorylation of 5’-cytosine and guanine...

NUCLEOTIDE METABOLISM

VI. THE PHOSPHORYLATION OF 5’-CYTOSINE AND GUANINE NUCLEOTIDES BY CELL FRACTIONS FROM RAT LIVER*

BY EDWARD HERBERTt AND VAN R. POTTER

(From the McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wisconsin)

(Received for publication, February 27, 1956)

The natural occurrence of guanine, cytosine, and uracil nucleotides in forms analogous to ADP’ and ATP (1-4)2 in the acid-soluble fraction of living t.issues makes it desirable to determine whether these nucleotides can substitute for adenine nucleotides in such reactions as oxidative phosphorylation, in addition to their role as part of the total nucleotide pool that serves as a source of specific coenzymes and structural units for the nucleic acids (1).

Recent work has established specific coenzyme functions for uracil (5, 6), cytosine (7), and guanine (8, 9) nucleotides. It has also been shown (10) that injected inorganic P32 is incorporated into the di- and triphosphates of all four nucleotides at nearly the same rates, suggesting that all the nucleotides might be receiving their terminal phosphates from a common donor. As the new nucleotides became available in quantity, their behavior in the reactions of oxidative phosphorylation was studied with isolated and recombined cell fractions from rat liver. It was reported (11) that rat liver mitochondria carrying on oxidative phosphorylation cannot phosphorylate UMP in marked contrast to AMP. However, UDP could be phosphorylated by the same system, and UMP could be phosphorylated by enzymes that occur elsewhere in the cell (11). With P32-labeled IP and ATP it was shown that the adenine nucleotides accept phosphate preferentially from oxidative phosphorylation donors and transfer phosphate to uracil nucleo-

* This work was supported in part by a grant (No. C-646) from the National Can- cer Institute, United States Public Health Service.

t Postdoctorate Research Fellow, National Heart Institute, National Institutes of Health, United States Public Health Service. Present address, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts.

1 The abbreviations used are AMP, ADP, and ATP, adenosine-5’.mono-, di-, and triphosphates; CMP, CDP, and CTP, cytidine-5’-mono-, di-, and triphosphates; UMP, UDP, and UTP, uridine-5’-mono-, di-, and triphosphates; GMP, GDP, and GTP, guanosine-5’-mono-, di-, and triphosphates; PCA, perchloric acid; IP, inorganic phosphate; SA, specific activity in counts per minutes (c.p.m.) per micromole; RNA, ribonucleic acid; DNA, deoxyribonucleic acid.

2 Communication from Dr. S. A. Morel1 of the Pabst Laboratories relating to iso- lation of CTP by Dr. S. H. Lipton, Dr. S. A. Morell, and Dr. A. Frieden.

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454 NUCLEOTIDE METABOLISM. VI

tides. The present study extends these findings to guanine and cytosine nucleotides.

This study and the previous reports (11, 12) have as their immediate ob- ject,ive the management of the various nucleotides in homogenate systems. It is assumed that such studies will be helpful in the long range objective of using homogenates and cell fractions to study RNA and DNA synthesis, adaptive enzyme formation, and similar phenomena.

EXPERIMENTAL

Cell Fractionation-For reasons to be discussed later, t’he ” cytoplasmic fraction” from rat liver cells was used in all of t’he enzyme studies reported here, except those in Table I and Fig. 6. The method of preparing bhis fraction has already been described in detail (11). It consists of the supernatant fluid and one wash solution obtained by centrifuging the nuclei at 600 X g.

Reaction Systems-The standard medium was made up in order that each 3.0 ml. of final reaction mixture would contain the following, the figures in parentheses representing the final concentrations: 15 pmoles of potassium pyruvate (0.005 M), 15 pmoles of potassium glutamate (0.005 M), 6 pmOleS

of potassium fumarate (0.002 M), 9 pmoles of MgC12 (0.003 M), 30 pmoles of potassium phosphate buffer at pH 7.2 (0.010 M), 160 equivalent mg. of cytoplasm, corresponding to the mg. wet weight of the liver, and sufficient solid sucrose to make the final concentration 0.25 M. The incubations were carried out at 30” as previously described (II), with details of nucleotide additions given in Table I or Figs. 1 to 6.

It should be pointed out that, instead of adding a single nucleotide or pair of nucleotides, it was more convenient and equally satisfactory to add the nucleotide preparations that were available, even though they contained more than one compound, and to determine the concentrations of each of the nucleoside or nucleotide products formed in the stated intervals of time, with a so called zero time control for each reaction mixture. This point was taken within less than 30 seconds after the reaction had started and temperature equilibrium had been reached. This procedure was preferred because in most reaction systems the use of any pair of nucleotides with dissimilar bases rapidly led to the production of both nu- cleosides and the mono-, di-, and triphosphates of each. However, with the analytical systems used, the concentration of each of the eight products was readily determined. The values at “zero time” are thus analytically determined and not assumed on the basis of what was added.

Analytical-The separation and quantitative analysis of the nucleotides in the supernatant liquids were accomplished by chromatography on Dowex 1 (X lo), 200 to 400 mesh anion exchange columns (3.0 cm. by 0.78 sq. cm.),


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E. HERBERT AND V. R. POTTER 455

mounted in glass columns (15 cm. long and sealed to Erlenmeyer flasks which were reservoirs for the eluent solutions). The preparation of the resin and the columns and the placement of the samples on the columns have already been described in detail (11). The procedure used here is a modi- fied version of that employed by Herbert et al. (11) for the separation of adenine and uracil nucleotides and by Hecht et al. (12) for the separation of adenine and cytosine ribose and deoxyribose nucleotides. Fig. 1 illustrates how elution with formic acid at pH 2 has been combined with ammonium formate elution at pH 5 to achieve the separation of the products of phosphorylation of CMP in the presence of the adenine nucleotides.3 The

TABLE I

Activity of Isolated and Recombined Cell Fractions in Phosphorylating Cytidine-5’-Monophosphate

In addition to the usual oxidative phosphorylation reactants each flask initially contained 3.0 ml. of incubation mixture with the following additions: 1.5 pmoles of CMP, 0.7 pmole of ATP, 10 pmoles of IP, and 160 equivalent mg. of the cell fractions. The incubation period was for 30 minutes. The cell fractionation procedure was performed as previously described (11). The Sz fraction is the supernatant fluid above the mitochondria and includes the microsomes and “fluffy layer.”

Cell components added

Mitochondria. .......................... St. ..................................... Mitochondria + Sx. ....................

“ + nuclei ..................

Whole homogenate. .....................

Net change in concentration of phosphate, cytidine, and cytosine nucleotides

IP 1 Cytidine / CDP 1 CTP

pswles &md3s pm&

-1.6 +0.10 0 +3.3 fO.90 +0.13 -4.7 0 $0.20 +0.5 +1.20 0 -3.5 +0.16 +0.25

pm&s

+0.10 0

+1.10 +0.13 t-o.9

composition of each peak in the chromatogram was determined in a manner described elsewhere (11). The chromatographic separation of adenine and guanine nucleotides was performed in the following way. After AMP wa.s eluted with 0.2 N HCOOH, six 5 ml. porbions of 1.5 N HCOOH followed by the same number of portions of 1.7 N HCOOH were used to elute GMP. ADP was eluted next with six 5 ml. portions of 2.5 N HCOOH followed by the same number of portions of 3.0 N HCOOH. GDP was eluted next with 8 to 10 ml. portions of 4.0 N HCOOH in 0.15 M ammonium formate. Then ATP was eluted with eight 5 ml. portions of 4.0 N HCOOH in 0.3 M ammonium formate and, finally, GTP with six 5 ml. portions of 4.0 N HCOOH

3 Another unknown compound which is formed in small quantities during the incubation is eluted by wash a solution (4 N HCOOH in 1 M Na formate) which is run through the column after the last eluent listed in the chromatogram in Fig. 1.


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456 SCCLEOTIDE METABOLISM. VI

in 0.4 M ammonium formate followed by six 5 ml. portions of 4.0 N HCOOH in 0.6 M ammonium formate. Thus techniques for separating the adenosine phosphates from any other series of ribonucleoside phosphates are available. Separations of more complex mixtures have not been attempted on a rout,ine basis.

51 15 MINUTES 00 . . - I J INCUBATION Q

n N :: - ,” 1.0 ;;

n 0 MINUTES i

8 a

INCUBATION fl P

FIG. 1. Chromatographic separation of the cytosine and adenine nucleotides and the distribution of radioactivity after 15 minutes incubation of the “cytoplasmic fraction” of rat liver with CMP and IP. The height of each step in the cross- hatched areas represents the counts per minute per ml. X lo-” in each fraction eluted from the column, while the height of the areas under the dark lines is the Exco. The eluents used are listed in the upper chart. The volume of each fraction was 10 ml. for water, 0.05 N HCOOH, 0.2 PI’ HCOOH, and 0.7 M AMF (ammonium formate), and 5 ml. for the remainder of the eluent,s. In addition to the standard components, each 3.0 ml. of the reaction mixture initially contained 2.85pmoles of CMP, 1.7 bmoles of ATP, and 26.4 pmoles of II’ containing 237,000 c.p.m. of I?. The volume of the

reaction mixture was initially 7.0 ml. The spaces between eluenk serve to empha- size that different solutions were used to elute each compound.

In addition to reading all the fractions eluted from the columns at 260 rnp in a model DU Beckman spectrophotometer, the peak tubes were also read at either 275 rnp or 278 ml.c. Since the adenine and cytosine nucleotides exhibit very different E&E260 ratios (1.9 to 2.0 for the cytosine nucleotides and 0.40 for the adenine nucleotides), these ratios mere used as an indication of the purity of the fractions. All fractions containing cytidine


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in some form were acidified to about pH 2 with concentrated HCI before reading. The quantities of the nucleotides in the column fractions were calculated by using molecular extinction coefficients of 13,700 for each of the cytosine nucleotides in acid solution at 278 rnp, 14,100 for each of the adenine nucleotides at 260 mp, and 12,700 for each of the guanine nucleotides at 260 mp (2). The numbers which appear in Figs. 1 to 6 and Table I as micromole quantities represent the nucleotide or IP content per 3.0 ml. of the final reaction mixture, obtained by adding up t,he values from all of the appropriate fractions.

The P32 used here was obt’ained in carrier-free form from the Oak Ridge n’ational Laboratory of t.he Atomic Energy Commission.* Radioactive counts were made directly on the samples as they were eluted from the columns with a Geiger-Miiller dip counter and a Berkeley decimal scaler.4 The calculation of the specific activities reported here is based on the aver- age of counts on at least four successive 5 ml. fractions. Only those fractions t’hat exhibited the proper E&E~~,J ratios were used in making these calculations. The cross-hatched areas in Fig. 1 show the distribution of radioactivity following a 15 minute incubation of 160 equivalent mg. of cytoplasm with IP32, CMP, and ATP. It is clear that there is a close cor- respondence between the E26a (height of the areas under the dark lines) and the distribution of t,he radioactivity.

Results

Phosph)oryZation of CMP-The chromatogram in Fig. 1 illust.rates the primary analytical data which form the basis of Figs. 2 to 6. It shows that following a 15 minute incubation of liver “cytoplasmic fraction” with CMP and ATP in the standard oxidative phosphorylation system at 30”, almost all of the CMP is converted to CDP and CTP. It should also be noted that once equilibrium is attained as it is here much larger amounts of CTP exist than CDP in agreement with the time-course studies to be reported later. The figure also demonstrates the oxidative uptake of inorganic P32 into ADP, ATP, CDP, and CTP, but, not AMP or CMP, which forms the basis of experiments reported later.

Localization of Enzymes That Phosphorylate Cytosine Nucleotides-Table I shows the result,s obtained wit,h various combinations of cell fractions, with CMP as the initial nucleotide. It. is clearly shown that mitochondria alone were unable to form either CDP or CTP and a slight breakdown to cytidine occurred. When mitochondria and the supernatant fluid from them (SJ were recombined the CMP was converted in high yield to CDP and CTP, while the S% fraction alone could form a small amount of CDP. These two

4 The counting was performed under the supervision of Dr. Charles Heidelberger to whom we acknowledge this valuable help.


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fractions together constitute the “cytoplasmic fraction,” which is here shown to be superior to the whole homogenate in terms of the extent of phosphorylation. This fact arises from the tendency of the nuclear fraction to dephosphorylate CMP to cytidine, as also shown by comparison of mitochondria and nuclei to mitochondria alone. In terms of the contributions of each fraction, nuclei dephosphorylate CMP, the Ss fraction converts CMP to CDP, and the mitochondria convert CDP but not CMP to CTP. These results parallel those obtained with UMP (11). It should be emphasized that in Table I the amount of ATP was insufficient to form 1.1 pmoles of CTP and 0.2 pmole of CDP in the absence of oxidative phosphorylation. This leads to the question of whether the phosphorylation of CMP and CDP can be directly coupled to oxidative processes or whether ATP inter- venes. This question could be partially answered by testing in the usual manner non-radioactive pools of t’he hypothetical intermediate, i.e. ATP, in diluting radioactive IP, in the course of its incorporation into CTP during oxidative phosphorylation. Secondly it could be attacked by determining whether labeled ATP could label CTP in the presence of non-labeled inorganic phosphate. Before proceeding to these experiments, the rapid interaction of the cytidine and adenine nucleot,ides in t,he oxidat,ive system was studied by non-radioactive methods.

Comparison between Time-Course of Phosphorylation of AMP and CMP- In Fig. 2 the time-course of the phosphorylation of CMP in the presence of added ATP (A and B) is compared with that of t’he phosphorylat.ion of AMP in t,he presence of added CTP (C and D). The general similarity of the t.wo cases can easily be seen. It should also be noted that wit.h the disappearance of CMP (A) and AMP (6) there is a corresponding decrease in ATP at 2 and 4 miuutes (B) and CTP (D) at 2 minutes, respectively. The fact that the theoretical amountIs of orthophosphate calculated from the observed changes in the nucleotides (dotted lines, A and C) are almost identical with t,he actually determined values at 2 and 4 minutes shows that transphosphorylation react,ions outpace oxidative phosphorylation during the period of the initial decreases in CTP and ATP.5 The data in Fig. 2 show that interaction among the cytosine and adenine nucleotides is very rapid and that they approach the same steady state regardless of whether the initial system cont,ains CMP and ATP or AMP and CTP.

Pa2 Studies on Pathway oj Incorporation of IPz2 into CTP-The following diagram represents a highly simplified version of two alternative pathways

5 It has frequently been noted as in the case above that the experimentally determined values of IP indicate a greater uptake of phosphate than the theoretical values in the later time periods. This may indicate that another unidentified phosphate compound accumulates in the later time period.


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L.-- TOTAL CYT. NUC. K---

* I A

MINUTES INCUBATION FIG. 2. Comparison of the time-course of phosphorylation of CMP with that of

AMP. A and R show the time-course of CMP phosphorylation in the presence of added ATP, while C and D show the time-course of AMP phosphorylation in the presence of added CTP. In addition to the standard components each 3.0 ml. of the reaction mixture contained the nucleotides shown. The initial incubation volume was 13.0 ml. in each case. The dotted lines show the inorganic phosphate values that may be calculated from the changes in the nucleotide balance.

that, might bc involved in the incorporation of 1P32 into CT!? during oxida- tivc phosphorylation.

1 Oxidative - ATP

Ip32 --) phosphorylation Primary 2

3 4 II

donor systems -.-- ----& CTP

Three kinds of experiments were carried out to determine which of the two pathways suggested in the diagram predominates in intact mitochon-


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dria. In the first kind of experiment three complete reaction mixtures mere prepared in duplicate with IP32 and mitochondria in each. Non- radioactive ATP was added to Flask 1, and non-radioactive CTP was added to Flask 2, and both were added to Flask 3. From the above diagram it is clear that the occurrence of Pathways 3 and 4 (cf. (Fig. 2)) should

2 0.2

F a d 0 2 4 6 8 cc MINUTES INCUBATION

FIG. 3. A comparison of the rate of exchange of IF with ATP and CTP. The specific activities of t.he terminal phosphates of the triphosphates and diphosphates are plotted relative to that of IP in Charts A and B, respectively. Curve 1 is from the incubation of IPa* with ATP, Curve 2 from incubation of IF with CTP, and Curve 3 from incubation of ATP and CTP in the same flask with W2. The di- and t.riphosphates lvere synthesized in the same flasks in which the experiment was carried out by preincubation for 12 minutes of 160 equivalent mg. of cytoplasm and oxidative substrate with AMP in Flask 1, CMP in Flask 2, and CMP and AMP in Flask 3. At zero time IP3* was added, and samples were removed successively at 2, 4, and 8 minute intervals. The total counts per minute accounted for as cytosine and adenine nucleotides and IP remained nearly constant at 2 and 4 minutes. In addition to the standard components, each 3.0 ml. of the final reaction mixture contained 0.85 pmole of ATP, 0.53 rmole of ADP, and 4.6 pmoles of IP containing 101,930 c.p.m. in Flask 1; 0.75 rmole of CTP, 0.45 pmole of CDP, and 4.4 Imoles of IP containing 108,000 c.p.m. in Flask 2 and 0.46 pmole of ADP, 0.74 pmole of ATP, 0.45 pmole of CDP, 0.79 pmole of CTP, and 7.9 pmoles of IP containing 128,000 c.p.m. in Flask 3.

diminish the radioactivity in both ATP and CTP in Flask 3 by simple dilution,‘j as compared with Flask 1 or 2. The occurrence of either Pathway 1 or Pathway 2 on a preferential basis should result in a greater radioactivity in the corresponding product. The data in Fig. 37 show that in

6 In none of the Ps2 experiments reported here did the monophosphates have de- tectable radioactivity.

7 The justification for plotting the results this \T--ay is given in a previous paper (11) in which it was shown that there is very close agreement between the SA of the


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E. HERBERT AND V. R. POTTER 4Gl

Flask 3 the ATP and CTP are both much less radioactive than when either nucleotide is present alone (Flasks 1 and 2), and in addition the initial labeling is preferential with respect to ATP, since at the 2 minute time point the ATP in Flask 3 is much more radioactive than the CTP. The crossing of the curves is due to the movement of P32 from ATP to CTP, and this phenomenon is utilized in another type of experiment described below. The data for the diphosphates show that in this case, also, the adenine nucleotide is labeled slightly preferentially. These data show that Pathway 2 is less important than Pathway 1.

Further evidence for the preferential occurrence of Pat~hmay 1 was obtained by a reaction system in which IP was not labeled and ATP was labeled

Oxidative zg===? ATPa IP~ phosphorylation

Primary 2 3 4

It donor systems -- CTP T-----

In this system the labeling of the CTP pool by the labeled ATP (Reaction 3) would compete with any direct labeling from non-radioactive inorganic phosphate (Reaction 2) that might occur. This system was set up by first preparing a complete reaction system containing both mitochondria and the supernatant fractions (cytoplasmic fraction) which is capable of phosphorylating AMP, ADP, CMP, and CDP, as shown earlier. To this were added IP32 and AMP. After 10 minutes of preincubation (not shown in Fig. 4) the flask contained labeled ADP and ATP, as shown at “zero time” in Fig. 4. At this t,ime a large pool of non-labeled IP was added to render the pool of IP relatively non-radioactive, and non-labeled CMP was also added. A sample was immediately taken to give the data shown at “zero time” in Fig. 4, when the SA of ATP was about 25 times that of the IP, and no CDP or CTP was present. Since the SA of CDP and CTP formed by 2 and 4 minutes are much higher than that of ATP and many times that of IP, it is clear that the rate along Pathway 3 greatly exceeds the rate of Pathway 2 under these conditions. That Pathway 1 is also rapid is shown by the rapid flow of non-labeled IP into ATP. The fact that the total counts per minute accounted for as cytosine and adenine nucleotides and IP remained almost constant at 2 and 4 minutes would indicate

terminal phosphate of UTP and ATP calculated by difference and those actually measured by determining the SA of ADP and UDP resulting from the partial hy- drolysis of the UTP and ATP samples. The results shown here are a valid measure of the Pz2 exchange, since the concentrations of IP, ATP, and CTP remained constant during the entire course of the incubations. The errors involved in the procedure have been treated statistically and reported (11).


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further that most of the P32 incorporated into CDP and CTP comes from ATP. Thus it appears that ATP turns over much more rapidly with respect to the large non-radioactive IP pool than CTP while transferring its P32 to CMP and CDP, i.e. Reactions 1 and 3 are much more rapid during CTP synthesis than 2 and 4.8

01 7 1 I I 2 4 8

MINUTES INCUBATION

FIG. 4. The incorporation of 1’32 into CDP and CTP during their formation from CMP in the presence of ATP3*. In addition to the usual components each 3.0 ml. of the reaction mixture initially contained 2.7 pmoles of CMP, 0.18 pmole of ATP with 9600 c.p.m., 0.27 pmole of ADP with 7600 c.p.m., and 41 rmoles of 11’ with 69,000 c.p.m. The counts per minute accounted for as cytosine and adenine nucleotides and IP are plotted as total at the top of the chart for each time interval. The initial incubation volume was 13.0 ml. The apparent increase in the total counts per minute is related to the small increase in the specific activity of a very large pool of inorganic phosphate.

A third type of experiment is shown in Fig. 5 which gives a comparison between the effect of a non-radioactive ATP pool on the rate of incorporation of IP32 into CDP and CTP during their synthesis from CMP (Chart A) and the effect of a non-radioactive pool of CTP of comparable size on the incorporation of IP32 into ADP and ATP during their synthesis from

8 When CTP was labeled with Pa2 and the synthesis of ADP and ATP from AMP was studied in a manner similar to that shown in Fig. 5, it was found that the terminal phosphate of ATP had lower SA than CTP at 2 and 4 minutes.


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AMP (Chart I?). The data plotted here are from the same experiment used to construct Fig. 2 in which the corresponding changes in the amounts of the nucleotides are shown. Chart B in Fig. 5 shows that at 2 minutes

1 0

TP= TERMINAL PHOSPHATE

A -

2 4’8 2 4’8

MINUTES INCUBATION

FIG. 5

I I I I 0 2 4 6 8 IO

MINUTES

FIG. 6

FIG. 5. The dilution effect of non-radioactive pools of ATP and CTP on the incorporation of IPa into the di- and triphosphates of cytidine and adenosine during their formation from CMP and AMP, respectively. The left-hand chart shows the effect of the non-radioactive pool of ATP on the incorporation of IPs2 into CDP and CTP (Experiment 1) and the right-hand chart the effect of non-radioactive CTP on similar incorporation in ADP and ATP (Experiment 2). In addition to the standard components each 3.0 ml. of the reaction mixture contained the nucleotides shown at time zero in Fig. 2. The counts per minute accounted for as the cytosine and adenine nucleotides and IP varied less than 5 per cent during the course of the incubation in both experiments. The initial incubation volumes were 13.0 ml. in both cases, and the total counts per minute were 500,000 per 3.0 ml. TP = terminal phosphate.

FIG. 6. A comparison between the effect of mitochondria on GMP (upper chart) and GDP (lower chart). In addition to the usual components, each 3.0 ml. of incubation mixture initially contained the nucleotides shown in both charts at zero time in one mixture.

the SA of the terminal phosphate of newly synthesized ATP is 3 times that of CDP and twice that of CTP. When the situation is reversed, i.e. synthesis of CTP and CDP from CMP is studied in the presence of non-radio-


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active ATP, the terminal phosphate of ATP has about the same SA as that of CTP and is higher than that of CDP at 2 minutes. Thus, under these conditions, the predominant pathway for incorporation of IP32 into ADP and ATP during synthesis from AMP does not involve t’he cytosine nucleotides as intermediates, but the adenine nucleotides appear to be on the main pathway of CDP and CTP synthesis from CMP.

Phosphorylation of Guanine Nucleotides-In view of the results with uracil (11) and cytosine nucleotides it was of interest to determine whether GMP and GDP would react like the pyrimidines or like adenine nucleotides. A sample of GDP containing a small amount of GMP was incu- bated with rat liver mitochondria and a mixture of ADP and AMP in the oxidative phosphorylation system -(Fig. 6). The GMP concentration remained constant, but the GDP was converted to GTP after a lag during which the ADP was converted to ATP. Thus it appears that GMP cannot be phosphorylated by mitochondria. Separate experiments showed that the supernatant fraction from mitochondria phosphorylates GMP. Further studies with P32 are needed to evaluate the acceptor role of GDP (9) in this type of preparation.

DISCUSSION

It is clear that the activities dealt with in this and the earlier reports (11, 12) are analogous to those of various extracts with “nucleoside monophosphate kinase” (13) and “nucleoside diphosphokinase” (14) activities plus the enzymes of oxidative phosphorylation. However in previous reports there does not appear to have been complete agreement as to the separation of the first two activities or as to their specificities. A calf liver extract would not catalyze the reaction between UTP and UMP, IMP, or CMP in the absence of adenine nucleotides (15, 16), while a yeast extract apparently was active in the absence of added adenine nucleotide (13). One yeast extract (13) would phosphorylate both mono- and diphosphates, while other reports dealt with an extract about which it was stated “nucleoside monophosphates do not participate in the reaction” (17, 18). These reports are of value in demonstrating the existence and properties of various types of enzyme activities and may help to interpret the present data, but they cannot possibly predict the present findings nor can they predict the proportional disposition of individual nucleotides among alternative pathways in whole tissues, or the partition of the various activities in various parts of the cell.

The present and previous reports (11, 12) show that rat liver mitochondria can phosphorylate any of the nucleoside diphosphates but lack nucleoside monophosphate kinases (13, 15, 16) or enzymes similar to adenylate kinase (19, 20) for phosphorylating the monophosphates other than AMP.


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The significant point is that the mitochondria do contain adenylaie kinase and that this enzyme does not act on CMP, UMP, or GMP in mito- chondrial preparations under any conditions studied.

These facts may help to elucidate cert’ain aspects of the biochemical organization of mitochondria and the rBle of adenine nucleotides in the regulation of oxidative phosphorylation (21-23). Recent studies by Sieke- vita and Potter (21, 22) on P32 incorporation and turnover suggest that adenylat)e kinase may be present at two sites in rat liver mitochondria. One site is in an “inner zone,” also the site of oxidative phosphorylation (21-23), and the other is in an “outer zone” which is in cont,act with the surrounding medium. These two “zones” are separated by some sort of permeability or enzymatic barrier, and the phosphorylated form of adenosine which most readily penetrates this barrier to the site of oxidative phosphorylation is AMP. Now if this barrier behaves in a similar manner toward the uracil, cytosine, and guanine nucleotides, then the phosphorylation of CDP, UDP, and GDP would occur chiefly in the “outer zone” and would occur by reaction with ATP rather than with donors in the hydrogen transport system, as the present work indicates. Furthermore, since mitochondria lack enzymes like adenylate kinase for converting UMP, CMP, and GMP to their diphosphate forms, it would follow that ADP would be the only phosphate acceptor of those tested that would be present in appreciable quantities at the site of oxidative phosphorylation in the inner zone. Consistent with this idea is the finding that the ratio of the amount of adenine nucleotides to the other kinds of nucleotides is much higher in the mitochondria than in the rest of the cell (21). The rate of oxidative phosphorylation then might depend upon the level of AMP in the “inner zone,” and this level in turn would depend upon shifts in the equilibrium of the reaction catalyzed by adenylate kinase in the ((outer zone”

AMP + ATP = 2ADP

in response to the rate at which ATP and ADP are used up in other parts of the cell. This does not mean that ADP would be the primary phosphate acceptor in all of the reactions of oxidative phosphorylation, because Sanadi et al. (9) have shown that GDP is a phosphate acceptor between succinyl- coenzyme A and ADP during the reduction of diphosphopyridine nucleotide with purified cu-ketoglutaric dehydrogenase.

The fact that ATP is present in such high concentration in the mitochondria also suggests that it is the adenine nucleotides that take part in the widest range of energy-requiring reactions of the cell. The large amounts of ATP formed in the mitochondria must be released into the supernatant solution (21-23) and then take part in transphosphorylations


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466 KUCLEOTIDE METABOLISM. VI

with the other nucleotides, while other parts of t.he cell maintain the r&e of utilizing the high energy compounds for nucleic acid synthesis and cer- tain coenzyme syntheses (24). The fact that enzymes for phosphorylating CMP, GMP, and UMP (11) to the diphosphates and UDP-glucose to UTP (24) exist in the supernatant fluid from mitochondria and nuclei is consistent with this idea.

Although the present findings with P32 indicate that the adenine nucleotides behave as preferential intermediates with respect to the uracil and cytosine nucleotides, they provide no basis for deciding whether or not they are obligatory intermediates.g It should be remembered that the results of P32 studies reported here represent the net effect of many reactions which are organized in the mitochondria to form the pattern of oxidative phosphorylation, and it is not surprising that, when t,he individual reactions that make up this pattern are studied, some reactions appear to be specific for individual nucleotides while others are satisfied by two or more different nucleotides (9, 22, 23, 25-27). These studies provide a foundation for further studies on more complex phenomena such as nucleic acid synthesis.

SUMMARY

It has been demonstrated that the “cytoplasmic fraction” from rat liver cells is capable of phosphorylating cytidine-5’-phosphate to cytidine di- and triphosphates during oxidative phosphorylation. As in the case of the uracil nucleotides, mitochondria have no effect on cytidine-5’-phosphate and guanosine-5’-phosphate, but will phosphorylate guanosine diphosphate to guanosine triphosphate. Rapid transphosphorylations have been shown to take place between the adenine and cytosine nucleotides. The results of experiments in which P32 was used as an indicator strongly suggest that adenosine triphosphate is an intermediate phosphate acceptor in the incorporation of inorganic phosphate into cytidine diphosphate and cytidine triphosphate during their formation from cytidined’-phosphate.

We would like to acknowledge the valuable technical assistance of Mrs. Heidi Haeberli who did a large share of the Beckman readings and P32 counting.

The authors are indebted to Dr. A. Frieden, Dr. S. A. Morell, and Dr. S. H. Lipton of the Pabst Laboratories for gifts of CTP and CMP and to Mr. Dan Broida of the Sigma Chemical Company for a gift of GDP.

8 The present data are in complete harmony with the interesting findings of Cooper, Devlin, and Lehninger (28) which appeared after the present report was submitted.


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Edward Herbert and Van R. PotterCELL FRACTIONS FROM RAT LIVER

AND GUANINE NUCLEOTIDES BYPHOSPHORYLATION OF 5'-CYTOSINE NUCLEOTIDE METABOLISM: VI. THE

1956, 222:453-467.J. Biol. Chem.

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nucleotide metabolism · nucleotide metabolism vi. the phosphorylation of 5’-cytosine and guanine...

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