Formation of 5'-AMP, 5'-GMP, 5'-CMP and 5'UMP was confirmed in isolated rat liver mitochondria incubated with alpha-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimidine nucleoside. Increased incorporation of 32Pi into ATP, GTP and UTP was observed by adding purine- and pyrimidine nucleosides. The phosphorylation of nucleosides was inhibited severely by arsenite and affected slightly by the addition of nuclear or post-mitochondrial fraction.</p

Inaba, Kozo

Oda, Takuzo

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Phosphorylation of purine and pyrimidine nucleosides by isolated rat liver mitochondria

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Acta Medica OkayamaVolume 29, Issue 5 1975 Article 5OCTOBER 1975Phosphorylation of purine and pyrimidinenucleosides by isolated rat liver mitochondriaKozo Inaba∗ Takuzo Oda†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Phosphorylation of purine and pyrimidinenucleosides by isolated rat liver mitochondria∗Kozo Inaba and Takuzo OdaAbstractFormation of 5’-AMP, 5’-GMP, 5’-CMP and 5’UMP was confirmed in isolated rat liver mito-chondria incubated with alpha-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimi-dine nucleoside. Increased incorporation of 32Pi into ATP, GTP and UTP was observed by addingpurine- and pyrimidine nucleosides. The phosphorylation of nucleosides was inhibited severelyby arsenite and affected slightly by the addition of nuclear or post-mitochondrial fraction.∗PMID: 132087 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 29, 367-375 (1975)PHOSPHORYLATION OF PURINE AND PYRIMIDINENUCLEOSIDES BY ISOLATED RAT LIVERMITOCHONDRIAKozo INABA* and Takuzo aDADepartment of Biochemistry, Cancer Institute, Okayama University Medical School,Okayama, JapanReceivedfor publication, May 9, 1975Abstract: Formation of 5'-AMP, 5'-GMP, 5'-eMP and 5'-UMPwas confirmed in isolated rat liver mitochondria incubated with cr-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimidinenucleoside. Increased incorporation of 32Pi into ATP, GTP and UTPwas observed by adding purine- and pyrimidine nucleosides. Thephosphorylation of nucleosides was inhibited severely by arsenite andaffected slightly by the addition of nuclear or post-mitochondrialfraction.There are few reports on the physicochemical analysis of RNA synthe-sized by isolated mitochondria since the rate of RNA synthesis in isolatedmitochondria is so low that this system can hardly be used for analysis. Itis, therefore, important to examine the incubation mixture system for RNAsynthesis of mitochondria. Purine- and pyrimidine nucleoside triphosphateshave been used as substrates of RNA synthesis in most cases of isolated mito-chondrial incubation. These nucleotides, however, are very poor substratessince the compartmentation of nucleotide transport in mitochondria (IJandthe transport of nucleotides into mitochondria are very restrictive. Swellingtreatment or prolonged incubation for removing the restriction of nucleotidetransport results in strong activation of latent ATPase.It is well known (2) that a2Pi is incorporated into purine- and pyrimi-dine nucleoside di- and triphosphates of endogenous nucleotides provided ratliver mitochondria are incubated with a-ketoglutarate and 32Pi, but the a-position of these nucleotides is not phosphorylated.The present paper reports that 32Pi is incorporated into the a-positionof purine- and pyrimidine nucleotides in isolated rat liver mitochondria aero-bically incubated with 32Pi in the presence of exogenous a-ketoglutarate andpurine- and pyrimidine nucleosides. An increased incorporation of32pi intoATP, GTP and UTP is observed by adding the corresponding nucleoside.* Present address: Department of Physiology, Okayama University Medical School3671Inaba and Oda: Phosphorylation of purine and pyrimidine nucleosides by isolatedProduced by The Berkeley Electronic Press, 1975368 K. INABA and T. ODAThese incorporations are free from the affect of nuclear or post-mitochondrialcontaminants.MATERIALS AND METHODSMaterials. Rats of the pure inbred Donryu strain were used. 32Pi (carrierfree) and 3H-uridine (1-3 Ci/mmole) were obtained from the Japan AtomicEnergy Institute and the Radiochemical Center, respectively. Purine- andpyrimidine nucleosides were products of Nakarai Pure Chemicals, Japan. Nuc-leotides and Dowex 1x 2 were purchased from Sigma Chemical Co.Preparation of mitochondria and incubation mixture. Mitochondria were isolatedfrom rat liver by a slight modification of the method of Hogeboom (3). Thepurified mitochondria (20-25 mg protein) were suspended in the incubationmedium if not stated otherwise. This purified mitochondria contained 170 mMsucrose, 5 mM Tris-HCl (pH 7.4), 2 mM MgCI 2 , 0.1 mM EDTA, 0.1 % bovineserum albumin, 10 mM potassium a-ketoglutarate, 2mM 32Pi (25,uCi/,umole,pH 7.4) and with or without purine- and pyrimidine nucleosides (4 mM adeno-sine, 1 mM guanosine, 2 mM cytidine and 2 mM uridine). The final volume ofthe incubation mixture was 10 ml. Incubation was carried out for 30 min at25°C under gentle shaking and stopped by placing the incubation flask in anice bath.Acid soluble fraction of mitochondria. The mitochondria were collected fromthe incubation mixture by centrifuging at 7,500xg for IOmin at O°C. To themitochondrial pellet 1 N perchloric acid was added at a final concentration of0.8N; the mixture was stirred with a glass rod for 20 min at O°C and centrifugedat 7,000 x g for 10 min. The supernatant was saved and the precipitate waswashed with a small volume of cold 0.8 N perchloric acid. This supernatantwas combined with the original one. The supernatant if not stated otherwisewas neutralized with 5 N potassium hydroxide and the precipitate was removedby centrifugation at 3,000 x g for 10 min.The 0.8 N perchloric acid-extract was heated in a boiling water bath for10 min before neutralization to detect the incorporation of 32Pi into the a-position of purine- and pyrimidine nucleoside di- and triphosphates.Ion exchange chromatography. The neutralized acid extract was applied to aDowex 1 x 2 formate form column (200-400 mesh, 0.8 cm x 25 cm). Elutionwas performed by passing into the 100 ml mixing chamber containing 50 ml ofdistilled water, the following in succession: (a) 30 ml of distilled water, (b)30ml of 1 N formic acid, (c) 240ml of 4N formic acid, (d) 150ml of 0.2M ammo-nium formate in 4 N formic acid, (e) 170 ml of 0.4 M ammonium formate in 4 Nformic acid and (f) 100 m1 of 1 M ammonium formate in 4 N formic acid. Theelution rate was 12ml/h and 3 ml fractions were collected. Eventhough in mostcases positive identification was accomplished by the coincidence of the radio-active peaks from the chromatographic fraction and the authentic marker, thecontents of radioactive peaks after lyophilization were chromatographed onpaper with appropriate solvents (4-6), and the radioactivity detected by radio-autography.2Acta Medica Okayama, Vol. 29 [1975], Iss. 5, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol29/iss5/5Phosphorylation of Nucleosides in Mitochondria 369Radioactivity. One ml of the fraction obtained from column chromato-graphy was pipetted to a planchet and dried under an infrared lamp. Measure-ments were performed with a gas-flow counter (Aloka Inc., Japan). In thecase of radioactive materials labeled with 3H, 0.1 ml of each fraction wasdissolved in 10 ml of Bray'S dioxane scintillator solution (7), and measurementswere performed with a Packard Tri-Carb Scintillation Counter (Model 3320)with external standardization.RESULTSRat liver mitochondria were incubated with 32Pi in the presence orabsence of exogenous ribonucleosides, as described in the section on Materialsand Methods. The acid soluble fraction of mitochondria after incubation wasapplied to a column of Dowex I X 2. The incorporation of 32Pi into ADP,ATP, GTP and UTP was detected in the endogenous incubation mixturesystem containing no external nucleosides (Fig. I).?CIO-2r---------------------------,1000500100Pi!AOP!ATP.!t'0~~ 10uoo:0oa:: 520015050 100Tube NumberFig. 1. Column chromatogram of the acid extract of rat liver mitochondria.Mitochondria were incubated aerobically with 32Pi at 25°C for 30 min in the absenceof external nucleosides. The acid extract was neutralized and applied to a DowexI x 2 (formate form) column with added markers, as shown on the figure. Elutionwas carried out as described in the text.The incorporation of 32Pi into UTP increased remarkably with theaddition of uridine in the incubation mixture (Fig. 2). The addition of adeno-sine, guanosine, cytidine and uridine resulted in increased incorporation of3Inaba and Oda: Phosphorylation of purine and pyrimidine nucleosides by isolatedProduced by The Berkeley Electronic Press, 197550o~~~~~----~':'--=----..,...----=--~---=....J100 150Tube NumberFig. 2. Column chromatogram of the acid extract of rat liver mitochon-dria. Mitochondria were incubated aerobically with 32Pi and uridine at 25°Cfor 30 min. The acid extract was treated as described in the legend of Fig. 1.370 K. INABA and T. aDAX10-11500Pi! AOP ATP100 ! ~E~~ ~oCl.N....,;.0?:' UTP:~ 10 !~0:0I:)Q: 5UTP~GTP!ATP~CTPGOP!XIO-l1000500100E AOP~ !~ 50Cl.&1~?:''> 10"£ 5'-GMPI:) !.2'tlI:)Q: ~or::::~~~---'--_---'::-"";'-- -.--- ---r_--"::::'::'-.J50 100 150 200Tube NumberFig. 3. Column chromatogram of the acid extract of rat liver mitochon-dria. Mitochondria were incubated aerobically with 32Pi, adenosine, guano-sine, cytidine and uridine at 25°C for 30 min. The acid extract was dealtwith as described in the legend of Fig. 1.4Acta Medica Okayama, Vol. 29 [1975], Iss. 5, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol29/iss5/5Phosphorylation of Nucleosides in Mitochondria 37132Pi into GMP, GDP, ATP and UTP (Fig. 3). The peaks of CDP and CTPoverlapped with those of Pi and ATP.The incorporation of 32Pi into purine and pyrimidine nucleotides wasinhibited considerably by the addition of sodium arsenite which is well knownas an inhibitor of a-ketoglutarate dehydrogenase (Fig. 4).XlcrZ.r---------------------------,10005100EQ.~50?::~ 10uo.2'Qoa:: 550Pi~AOP!Fig.. 4. Column chromatogram of the acid extract of rat liver mitochon-dria. Mitochondria were incubated aerobically with 32Pi, adenosine, guano-sine, cytidine and uridine in the presence of 2 mM sodium arsenite at 25°C for3Omin. The acid extract was dealt with as described in the legend of Fig. 1.To detect the incorporation of 32Pi into the a-position of purine- andpyrimidine nucleoside di- and ..triphosphates, after incubation with 32Pi,0.8 N perchloric acid extract from the mitochondria was heated in a boilingwater bath for 10 min. After neutralization and centrifugation, the heattreated-acid extract was applied to a column of Dowex I x 2. The chroma-togram is shown in Fig. 5.CMP, AMP and GMP were detected by the radioactive peaks in thechromatographic fraction and by paper chromatography in the heat treated-acid extract from mitochondria incubated with 32pi, adenosine, guanosine,cytidine and uridine (closed circles) in contrast to the endogenous incubationmixture system (open circles). In the formation of eMP by mitochondria,cytidine was required and could not be replaced by uridine (triangles).5Inaba and Oda: Phosphorylation of purine and pyrimidine nucleosides by isolatedProduced by The Berkeley Electronic Press, 19753721000EDo~500ll.:::'0 400'~.;'t; 300co~ca:: 20010K.INABA and T. 0DA20 30Tube Number405'-GMP~~Fig.5. Column chromatogram of the heat treated-acid extract ofrat liver mitochondria. Mitochondria were incubated aerobically with32Pi in the presence and absence of external nucleosides. The acidextract was heated in a boiling water bath for 10 min and dealt withas described in the legend of Fig. I. 0-0, without external nucleo-side; A······A, with adenosine, guanosine and uridine; e--e,with adenosine, guanosine, cytidine and uridine.Since the peaks of UMP and 112Pi overlapped on the chromatogram,mitochondria were incubated with 3H-uridine and Pi. The chromatogram ofthe heat treated-acid extract from mitochondria after incubation is shown inFig. 6. Two peaks were detected besides the first bulk peak of 3H-uridine.The second (or middle) peak has not yet been identified. The last peak wasidentified as UMP by the elution position and paper chromatography.It is important to determine whether the contamination of nuclear orpost-mitochondrial fraction in the mitochondrial preparation affects the phos-phorylation of purine- and pyrimidine nucleosides. Mitochondria were incu-bated with 32Pi and purine- and pyrimidine nucleosides or with 3H-uridineand Pi with and without the addition of nuclear or post-mitochondrial frac-tion. It was clear that both the nuclear and post-mitochondrial fractions donot significantly affect the phosphorylation of purine- and pyrimidine nucleo-sides with mitochondria (Table I).6Acta Medica Okayama, Vol. 29 [1975], Iss. 5, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol29/iss5/5Phosphorylation of Nucleosides in Mitochondria 3735'-UMP.~X10-'r---------------------------------,1000100106:I:..'0 4>.:Eu00:00a:: 2oL..------,------..,------r--------r-----'403010 20Tube NumberFig.6. Column chromatogram of the heat treated-acid extract of rat livermitochondria. Mitochondria (2 mg protein) were incubated aerobically at25°C in the incubation medium containing 3H-uridine (20,uCi/m,umole/ml) for30 min. The acid extract was heated in a boiling water bath for 10 min andapplied to a Dowex I X 2 (formate form) column with authentic markers afterneutralization. Elution was carried out by passing into the 100 ml mixingchamber containing 50 ml of distilled water the following in succession: (al30 ml of distilled water, (b) 30 ml of 1 N formic acid and (c) 70 ml of 0.2 Mammonium formate in 4 N formic acid.T'ABLE I EFFECT OF NUCLEAR AND POST-MITOCHONDRIAL FRACTIONS ON NUCLEOSIDEPHOSPHORYLATION IN MITOCHONDRIAFraction added to incubation CMP AMP GMP UMP*mixture (%) (%) (%) (%)None 100 100 100 100Nuclear fraction** 106 97 95 92Post-mitochondrial fraction*** 106 96 97 90* Rat liver mitochondria were incubated with 3H-uridine and Pi under the sameconditions described in the legend of Fig. 6.** Nuclear fraction (7 mg protein) obtained from the mitochondrial isolation process.*** Post-mitochondrial fraction (7 mg protein) obtained from the supernatant afterseparation of mitochondria.Rat liver mitochondria were incubated aerobically at 25°C for 30 min with 32pi, adeno-sine, guanosine, cytidine, and uridine or 3H-uridine plus Pi, in the presence or absence ofa nuclear or post-mitochondrial fraction. The details are described in Fig. 5 and Fig. 6.7Inaba and Oda: Phosphorylation of purine and pyrimidine nucleosides by isolatedProduced by The Berkeley Electronic Press, 1975374 K. INABA and T. ODADISCUSSIONIt has been reported (2) that 32Pi is incorporated into purine- and pyri-midine nucleoside di- and triphosphates, when rat liver mitochondria wereincubated with a-ketoglutarate and 32Pi. The present results show thatthe a-position of the nucleotides was not phosphorylated significantly in theabsence of external nucleosides, but by adding purine- and pyrimidinenucleosides, the a-position of the corresponding nucleotides was phosphory-lated in coupling with a-ketoglutarate linked phosphorylation. The increasedincorporation of 32Pi into ATP, GTP and UTP was also observed by addingnucleosides. The phosphorylation of nucleosides was free from the effects ofnuclear or post-mitochondrial contaminants.There are differences in the results of Tsiftsoglou and Geogratsos (8) andthe present study on nucleotide formation in mitochondria. These investigators(8) reported that 3H-cytidine nucleotide was formed when mouse liver mito-chondria were incubated for a prolonged period at 37°C with 3H-uridine,ATP, succinate and malate. According to their data, the peaks of labelednucleoside monophosphate were higher than those of the triphosphates in theacid extract of the mitochondria. The present results indicate that externalcytidine is essentially required for the formation of CMP. Furthermore, peaksof labeled nucleoside 5'-monophosphates in the acid extract of mitochondriawere very small in contrast to those of nucleoside triphosphates when the acidextract was not heated in a boiling water bath for 10 min. These resultsmay be due to differences in the incubation mixture systems. However, thereis the possibility of contaminants in their mitochondrial preparation system (8)since the incubation was carried out for a relatively long time at 37°C andthe oxidative phosphorylation would be uncoupled.What is the most likely pathway for the incorporation of 32Pi into thea-position of purine- and pyrimidine nucleotides in mitochondria? The mostprobable one seems to be a ., salvage pathway." It involves the participationsof purine- and pyrimidine nucleoside kinases that are not well defined, coupledwith a-ketoglutarate linked-phosphorylation. There is no evidence for thepresence of an enzyme in de no)o synthesis of purine- and pyrimidine nucleoside-5'-phosphates in mitochondria. The incorporation of :l2Pi into adenosine-2'(3')-phosphate observed in the heat treated-acid extract seems to derive from thedegradation of NADP formed by the phosphorylation of endogenous NAD.It is very important to study the physicochemical properties of RNAsynthesized in the mitochondrial incubation mixture system. We have alreadyobserved that 32Pi is further incorporated into mtRNA in the mitochondrialincubation mixture system. The details will be reported elsewhere.8Acta Medica Okayama, Vol. 29 [1975], Iss. 5, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol29/iss5/5Phosphorylation of Nucleosides in Mitochondria 375Acknowledgment: This work was supported in part by a Grant-in-Aid for Scientific Re-search from the Japanese Ministry of Education.REFERENCES1. Klingenberg, M. and Pfaff, E.: Structural and functional compartmentation in mito-chondria. In .. B. B. A.Library, "ed. J. M. Tager, S. Papa, E. Quagliariello and E. C. Slater,Elsevier Publishing Company, Amsterdam, London and New York, Vol. 7, p.I80, 1966.2. Heldt, H. W.: The participation of endogenous nucleotides in mitochondrial phosphate-transfer reactions. In" B. B. A. Library," ed. J. M. Tager, S. Papa, E. Quagliariello andE. C. Slater, Elsevier Publishing Company, Amsterdam, London and New York, Vol. 7,p.51, 1966.3. Hogeboom, G. H.: Fractionation of cell components of animal tissues. In Methods inEnzymology, ed. S.P. Colowick and N.O. Kaplan, Academic Press, New York, Vol. I, p.16,1955.4. Tsuboi, K. K. and Price, T. D.: Isolation, detection and measure of microgram quanti-ties of labeled tissue nucleosides. Arch. Biochem. Biophys. 81, 223, 1959.5. Magasanik, B., Visher, E., Doniger, R., Elson, D. and Chargaff, E.: Separation andestimation of ribonucleosides. J. Biol. Chem. 186, 37, 1950.6. Markham, R. and Smith, J. D.: Nucleic acids (IV) of the turnip yellow mosaic virus-of the tomato bushy stunt virus-filter outfit for the short-wave ultra-violet. Biochem. J.49, 401, 1950.7. Bray, G. A.: Liquid scintillator for counting aqueous solutions. Anal. Biochem. 1, 276,1960.8. Tsiftsoglou, A. S. and Geogratsos, J. G.: Metabolism of nucleosides by isolated mouseliver mitochondria. Biochim. Biophys. Acta 262, 239, 1972.9Inaba and Oda: Phosphorylation of purine and pyrimidine nucleosides by isolatedProduced by The Berkeley Electronic Press, 1975

Fractionation of cell components of animal tissues.

Isolation, detection and measure of microgram quantities of labeled tissue nucleosides.

Liquid scintillator for counting aqueous solutions.

Metabolism of nucleosides by isolated mouse liver mitochondria.

Nucleic acids (IV) of the turnip yellow mosaic virusof the tomato bushy stunt virus-filter outfit for the short-wave ultra-violet.

Separation and estimation of ribonucleosides.

Structural and functional compartmentation in mitochondria.

The participation of endogenous nucleotides in mitochondrial phosphatetransfer reactions.

Phosphorylation of purine and pyrimidine nucleosides by isolated rat liver mitochondria

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