a) A modified procedure of the WIDNELL and TATA8 method yields rat liver nuclei manifesting a high degree of purity and activity. b) These nuclei contain a nucleoside-dependent phosphorylating activity that is readily released and apparently unrelated to either glycolysis or respiration. c) The main incorporation of the 32Pi is into ribose-I-phosphate; nucleoside phosphorylase activity
satisfactorily accounts for the observed purine nucleoside stimulation of the nuclear phosphorus metabolism.</p

Utsumi, Kozo

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Nucleoside-dependent synthesis of organic phosphorus compounds by rat liver nuclei

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 21, Issue 5 1967 Article 2OCTOBER 1967Nucleoside-dependent synthesis of organicphosphorus compounds by rat liver nucleiKozo Utsumi∗∗University of California,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Nucleoside-dependent synthesis of organicphosphorus compounds by rat liver nuclei∗Kozo UtsumiAbstracta) A modified procedure of the WIDNELL and TATA8 method yields rat liver nuclei man-ifesting a high degree of purity and activity. b) These nuclei contain a nucleoside-dependentphosphorylating activity that is readily released and apparently unrelated to either glycolysis orrespiration. c) The main incorporation of the 32Pi is into ribose-I-phosphate; nucleoside phospho-rylase activity satisfactorily accounts for the observed purine nucleoside stimulation of the nuclearphosphorus metabolism.∗PMID: 4232094 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 21 207-211 (1967)NUCLEOSIDE-DEPENDENT SYNTHESIS OF ORGANICPHOSPHOROUS COMPOUNDS BY RAT LIVER NUCLEI*Kozo UTSUMI**Department of Physiology, University of California,Berkeley, California, U. S. A.Received for publication, August 19, 1967Since the initial discovery of OSAWA, ALLFREY and MIRSKyl that calf thymusnuclei in vitro synthesize ATP from endogenous ADP and AMP in an oxygen-dependent process, the mechanism of this ATP formation. has been activelysought. Also the distribution of this activity in other nuclei as in rat liverhas also been investigated. Although it is clear that ATP formation in ratthymus is indeed oxygen-dependent, it is also clear from the studies of McEWEN,ALLFREY and MIRSKy2 and BETEL and KLOUWEN3 that the mechanism of thisprocess differs from oxidative phosphorylation in mitochondria. By contrast tothymus nuclei, investigations of ATP formation by rat liver nuclei have metwith considerable difficulty. One problem has been the isolation of fresh intactrat liver nuclei in a high state of purity. Hence, earlier results suggesting ATPsynthesis were subject to the criticism that they reflected extranuclear contamina-tion.PENNIALL, Lm and SAUNDARS4 and PENNIALL, SAUNDERS and Lm5 havereported an active incorporation of 32P-Iabeled inorganic phosphate (32PJ into acid-soluble phosphorus compounds. The synthesis of these acid-soluble phosphoruscompounds is oxygen-dependent and incorporation was observed to be enhancedin the presence of adenosine and guanosine6• BETEL and KLOUWENs haveobserved aerobic synthesis of ATP by aged rat thymus nuclei to be stimulatedby inosine and adenosine. This nucleoside stimulation of nuclear phosphorylationhas also been confirmed by BASHIRELAHI and BALLAM7 who specifically reportedthat the ATP synthesis in rat thymus nuclei was enhanced under these condi-tionE'. In view of this, it seemed worthwhile to investigate further the occurrenceof this enzyme system in isolated and highly purified rat liver nuclei in order todetermine optimum conditions for incorporation, and to identify the acid-solubleorganic phosphorus compounds synthesized.* This research was supported by Cancer Research Funds of the University of California.** On leave from the Department of Biochemistry, Cancer Institute, Okayama UniversityMedical School, Okayama, Japan.2071Utsumi: Nucleoside-dependent synthesis of organic phosphorus compounds byProduced by The Berkeley Electronic Press, 1967208 K. UTSUMIMATERIALS AND METHODSAfter an extensive evaluation of existing methods for isolation of ratliver nuclei, the method of WIDNELL and TATA8 involving sucrose densitygradient fractionation was adopted. In the final step of this procedure thenuclear pellet, designated N1) forms a clear supernatant (SJ) below. This nuclearfraction was further purified by centrifugation through a IO-fold volume of 0.25M sucrose to yield another supernatant (S2) and upon recentrifugation underthese conditions a final nuclear (N2) fraction was obtained. The N2 fraction iscomposed of highly purified intact nuclei based upon the following criteria: a)electron microscopy reveals virtually no contamination by other particles; b)RNA: DNA ratio decreased 10 per cent as compared to nuclei isolated by themethod of WIDENELL and TATA; c) glucose-6-phosphatase and NAD pyrophos-phorylase of 177 and 105 m/J.moles/mg nuclear protein/I20 min respectively.Glucose-6-phosphatase activity was about half and NAD pyrophosphorylase was30 per cent higher than found by WIDNELL and TATA for the N1 fraction.For the estimation of 32Pi incorporation into organic phosphorus compounds,nuclei were incubated in 0.25M sucrose, 5.0mM Tris-HCI buffer (pH 7.1),3.0 mM MgCI2, 1. O,t..tc carrier-free 32pi, and 2.0 mM inosine (when present) ina final volume of 2. 5 ml. The reaction was stopped by adding 5.0 ml of O. 6 MNa2S03 in 0.91\-:1 HCI04, and then carrier phosphate (1.0 mM as sodium salt) wasadded. Organic phosphorus compounds were separated from inorganic phosphateby the column chromatographic method of HAGIHARA and LARDy9 and thenradioactivity of the organic phosphorus compounds was determined.Separation of individual phosphorus compounds was made using Dowex-I-formate according to the column chromatographic method of SIEKEVITZ andPOTTER'o as modified by TERADAll• Ribose-I-phosphate was identified by paperchromatography according to MORTIMER12• Protein was determined by the methodof LOWRY, ROSEBROUGH, FARR and RANDALL13• Chemicals were obtained com-mercially; nucleosides were obtained from Sigma Chemical Co., St. Louis,Mo.RESULTSThe phophorylation activities of the NJ, N2, SJ and S2 fractions were ex-amined by testing for the formation of S2P-organic phosphorus compounds.Typical results are given in Table 1 for controls and fractions supplementedwith inosine. By comparing the inosine effect on Sl with that on S2 one wouldpredict a similar degree of stimulation if the effect was due to extranuclearenzymes. However, the result shows that the inosine stimulation of nuclearphosphorylation is not due to a cell sap component but rather to a loosely bound2Acta Medica Okayama, Vol. 21 [1967], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol21/iss5/2Nucleoside-Dependent Nuclear PhosphorylationTaIJ]e 1 Inosine SlilJ1u!;Jlion of 32Pi Incorporation into Acid-soluble OrganicPhosphorus Compounds by l~at Liver NucleiTotal Specific RelativeFraction* incorporation activity inosine(cpm) (cpm/mg protein) effectNl 3,920 512Nl+lnosine 10,460 1,366 2.7xSI 3,380 4,500Sl+Inosine 4,980 6,640 1.5xN2 1,890 242N2 + Inosine 2,700 346 lAXS2 645 2, 790S2+Inosine 7,350 31,100 10.3X209* The reaction mixture was incubated aerobically with shaking at 25°C for 30 min as describedin the methods employing 0.6 ml of each fraction.component of the nuclear fraction which is significantly released into the ~ frac-tion upon washing the N 1 fraction. This interpretation is corroborated by thefinding that after washing N 1 twice the inosine stimulation is still retained by theN:1 fraction. The time course of inosine stimulation of 32Pj incorporation intoorganic phosphorus compounds of N2 proceeds in a linear fashion over a 60·minassay interval.A number of inhibitors were tested on the inosine-dependent phosphoryla-tion. The patterns observed did not offer clear results which might suggest arole for glycolysis or of an electron transport type of system in the measuredactivities. For example, the inhibition of activity was only 19 per cent foriodoacetate and antimycin A, 11 per cent for 2, <1 -dinitrophenol when tested atconcentrations which check glycolysis or respiration.Since it had already been reported6 that guanosine stimulates nuclear phos-phorylation, parallel experiments were made to compare effects of inosine andguanosine. Figure 1 illustrates the type of elution pattern obtained for thelabeled components of the acid-soluble extract. In this experiment, a 30-secincubation in a nucleoside-free reaction medium was examined. Incubation ofthe N2 fraction under aerobic conditions even for a 30-sec interval is sufficient todemonstrate incorporation of 32Pi into organic phosphorus compounds; e. g. it isclear that significant radioactivity has already found its way into the ATP andribose-I-phosphate fractions and some radioactivity is indicated in the ADP frac-tions and in certan unidentified peaks. Figure 2 compares the relative amountsof the identified substances for controls, and for the nucleoside-containing reac-tion mixtures. Both nucleosides may act similarly since both stimulate theribose-I-phosphate formation. It is also evident from Fig. 2 that the decay in3Utsumi: Nucleoside-dependent synthesis of organic phosphorus compounds byProduced by The Berkeley Electronic Press, 1967210 K. UTSUMI60 minInosine Guanosine60 minC:::J_D_Jeooo CPM1»1-30 sec 60 minControls~ t~_· JRoioP~~160ATPj80 120Tube numberRoioPj401412~ 100-Ug~:~va0.:.0a""Fig. 1 Organic phosphorus compounds identifiedin the acid-soluble extract of the rat liver nuclearfraction.The reaction mixture contained 48 mg protein ofthe Nz fraction and other conditions as in Table 1,in a final volume of 12 mJ. Incubation was for a30-sec period at O°C in the absence of nucleosideand the reaction was stopped by adding ice-coldHCl04 to a final concentration of 0.5 N. AMP,ADP and ATP were added to the extract to facili-tate identification.Fig. 2 3ZPi incorporation into adeninenucleotides and ribose-I-phosphate by thenuclear (Nz) fraction.Conditions as in Fig. 1 and Table 1.Controls were incubated for 30 sec at O°Cand for 60 min at 25°C. Experimentalswere incubated for 60 min at 25°C in thepresence of nucleosides (either 20 pmolesof inosine or 2 rmoles of guanosine per12 ml of reaction mixture). The relativeamounts of labeled compounds were esti-mated from the area of the elution peaks.the 32P-ATP peak that occurs during incubation is paralleled by an increase inthe 32P_ADP component, suggesting an interconversion of ADP and ATP.COMMENTInosine is at a U cross-road" for pathways of nucleotide metabolism and so itwould be expected to promote nuclear phosphorylation. In other experimentsthe N2 fraction has been observed to demonstrate an active inosine-dependenthypoxanthine formation. This data concerning net inosine stimulation of 32Pi in-corporation into the acid-soluble fraction by rat liver nuclei show a similar effectfor net increase of ATP synthesis as was reported for rat thymus nuclei byBETEL and KLOUWEN9• Fractionation of the acid-soluble organic phosphorus com-pounds shows that 32P_ATP is synthesized by incubation at O°C for 30 sec, andshows a similar elution pattern of nucleotides to that found by OSAWA et all inrapidly isolated calf thymus nuclei. After continued incubation, however, the4Acta Medica Okayama, Vol. 21 [1967], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol21/iss5/2Nucleoside-Dependent Nuclear Phosphorylation 21132P-ATP-level decreases, an effect which might reflect ATPase and adenylate-kinase activities and/or ATP regulation of other unknown pathways as is sug-gested by the simultaneous rise in the ~2p_ADP peak. In the presence of inosineor guanosine the 32P_ADP levels are slightly higher than in the control incubatedunder identical conditions except for the addition of nucleoside This may in-dicate that the tested nucleosides stimulate ADP formation by an undeterminedpathway. The main effect of added i1Ucleosides is in promoting 32Pi incorpora-tion. This might be attributed to a (purine) nucleoside phosphorylase since themain labeled peak was identified as ribose-I-phosphate. Hence, a significantportion of the nuclear phosphorylating activity does not appear as nucleosidephosphorylation and is channelled into other pathways having ribose-I-phosphateas intermediate. Therefore, the action of added nucleoside in rat liver nucleidiffers from that observed in thymus nuclei.SUMMARYa) A modified procedure of the WIDNELL and TATA8 method yields rat livernuclei manifesting a high degree of purity and activity. b) These nuclei containa nucleoside-dependent phosphorylating activity that is readily released andapparently unrelated to either glycolysis or respiration. c) The main incorpora-tion of the 32Pi is into ribose-I-phosphate; nucleoside phosphorylase activitysatisfactorily accounts for the observed purine nucleoside stimulation of thenuclear phosphorus metabolism.ACKNOWLEDGEMENTThe author expresses his thanks to Prof. LESTER PACKER for helpful sug&estions, and toMIGUEL LLINAS for assistance. The award of a Research Travel Grant from the JapaneseMinistry of Education is gratefully acknowledged.REFERENCES1. OSAWA, S., ALLFREY, V.G. and MIRSKY, A.E.: ]. Gen. Physiol. 40, 491, 19572. McEWEN, B. S., ALLFREY, V. G. and MIRSKY, A. E.: ]. BioI. Chern. 238, 2579, 19633. BETEL, 1. and KLOUWEN, H. M.: Bioclzirn. Biophys. Acta 131, 453, 19674. PENNIALL, R., LIU, S. -M. and SAUNDERS, J. P.: Biochirn. Biophys. Acta 76, 170,19635. PENNIALL, R., SAUNDERS, ]. P. and LIU, S. -M.: Biochemistry 3, 1454, 19646. PENNIALL, R., SAUNDERS, J. P.: Biochemistry 3, 1459, 19647. BASHIRELAHI, N. and DALLAM, R. D.: Federation Proc. 25, 530, 19668. WIDNELL, C. C. and TATA, ]. R.: Biochem. ]. 92, 313, 19649. HAGIHARA, B. and LARDY, H. A.: ]. BioI. Chern. 235, 889, 196D10. SIEKEVITZ, P. and POTTER, V.R.: ]. BioI. Chem. 215, 221, 195511. TERADA, S.: ]. lap. Biochem. Soc. 31, 795, 195912. MORTIMER, D. C.: Can. ]. Chern. 30, 653, 195213. LOWRY, D. H., RO':EBROUGH, N. J., FARR, A. L. and RANDALL, R. J.: ]. BioI. Chern.193, 265, 19515Utsumi: Nucleoside-dependent synthesis of organic phosphorus compounds byProduced by The Berkeley Electronic Press, 1967

Nucleoside-dependent synthesis of organic phosphorus compounds by rat liver nuclei

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