The Identification of 2-Dimethylamino-6-hydroxypurine and Its Ribonucleoside in Urine of Normal and Leukemic Subjects

8/4/2019 The Identification of 2-Dimethylamino-6-hydroxypurine and Its Ribonucleoside in Urine of Normal and Leukemic Su

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1963;23:1824-1829. Published online December 1, 1963.Cancer ResKay Fink, William S. Adams, Frances W. Davis, et al.Ribonucleoside in Urine of Normal and Leukemic SubjectsThe Identification of 2-Dimethylamino-6-hydroxypurine and Its

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The Iden tification o f 2 -D ime thylamino-6 -hyd roxypu rineand Its Ribonucleoside in Urine of Normal andLeukem ic Subjects*

KAY FINK,W ILLIAMS. ADAMS,FRANCESW. DAVIS,ANDMISAENAKATANI(Depar tment s o f Medi ci ne and B ioph ys ic s, S choo l o f Medi ci ne , Un iv er si ty o f Ca li fo rn ia a t L osAngeles ; and Wadswor th Hospi ta l, Ve te rans Admin ist ra t ion , Los Angeles , Ca li fo rnia' )

SUMMARYU rine from norm al and leukem ic subjects on controlled low -purine diets w as fractionated by ion-exchange colum n chrom atography, and the various effluent peaksexhibiting absorbance at 260 m/i were subjected to two-dimensional filter paperchrom atography for further resolution. It appeared from gross exam ination of thec hrom atogram s that there w ere a greater num ber and c oncentratio n of blue flu orescentsubstances in m uch of the leukem ic urine. Tw o of these com pounds have been identif ie d a s 2 -d ime th yl am in o-6 -h yd ro xy pu ri ne and its ri bo nu cle os id e.

A number of methylated purines have beenfound in hum an urine by W eissm ann, Brom berg,and G utm an (33). These include 1-m ethylhypo-x an th in e, 7 -me th ylg uanin e, 8 -h yd ro xy -7 -me th yl-guan in e, 1 -me thylguanine , and 6 -hyd roxy -2 -me th -y lamin op urin e. T he ir p re se nc e h as b ee n c on firm edby Park et al. (24), who also reported increasedexc re tio n o f 1 -me th ylh yp ox an th in e, 7 -me th ylg uan in e, a nd 8 -h yd ro xy -7 -m eth ylg ua nin e in a n umbe rof leukem ic patients. In a continuation of a studyof urinary pyrim idines and purines in norm al andle uk em ic u rin es b y a c ombin atio n o f io n-e xc ha ng eand filter paper chromatography (1), a greaternum ber and concentration of blue fluore scent substances were observed in much of the leukemicurine. Tw o of these com pounds have been identifie d a s 2 -d im eth ylamin o-6 -h yd ro xy pu rin e a nd itsribonucleo side. E lion et al. (13) have synthe sized2 -d ime th ylam in o-6 -h yd ro xy pu rin e, a nd its b io lo gical occurrence as a minor base in RNA has beenreported by Smith and Dunn (29).MATERIALS AND METHODSN orm al subjects and leukem ic patients of various types were maintained on a controlled low-

* T h is re sea rch w as su pp orte d b y G ran t CA-0 24 33 , U nite dStates Public H ealth S ervice, and by G rant P-813 from theAme ri ca n Canc er So ci et y.R ec eiv ed f or p ub li ca tio n J ul y 2 7, 1 96 3.

purine diet for 5 days prior to the collection of a24-hour urine specimen. A liquots of the urinew ere su bje cte d to io n-e xc ha ng e c hroma to gra ph yby m odification of a procedure described earlier(1). The urine was added to a column of Dowex2-chloride (X 8, 200-400 m esh) ani n exchangeresin, and gradient elution was accom plished bythe gradual delivery of a solution of 0.1 M aceticacid an d 0.025 M ammonium chloride into a m ixingc hamb er c on ta in in g a c on sta nt v olume o f so lu tio n,w hic h initially consisted o f 0 .22 M ammonium hydro xide and 0 .025 M ammon ium chlorid e. A grad ual fall in pH from 10.4 to about 7.5 w as achieved,and the eluate from the column was collected in2.5-m l. fractions. In m ost cases a volum e of urineequivalent to 6 m g. of creatinine was applied to acolumn 24 X 0.75 cm., and the volum e in the mixing cham ber w as 250 m l.Some urine was also fractionated on a largerscale, w ith 75 per cent of a 24-hour volum e, by thefo llowin g p ro ce du re : O ne -fo urth o f a to ta l 2 4-h ou rc olle ctio n o f u rin e w as re du ce d in v ac uo to a pp ro ximately 50 ml. To this was added an equal volum eof solution #1 (0.6 gm . ammonium formate and15.0 m l. o f conce ntrated ammonium hydrox ide perliter), the pH of the mixture was adjusted to 10.4with additional am monium hydroxide, and afterfiltration the m aterial was added to a colum n (46X 2.5 cm .) of D ow ex 2-form ate re sin ( X 8 , 200-40 0

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F INK e t a l. U r in a r y 2 -D ime t h ylam i n o-6 -h y d r ox ypu r i n e 1825m esh). T he am monium form ate solution jf l w asem ploy ed as the inf luent until 40 f ractions of25 m l. each of ef f luent had been collected. Cre-at in ine and N-me thy l- 2- py ridone -5 -carboxamidew ere thus rem ov ed. A t this stage solution #2(0.6 gm . amm onium f orm ate and 3.8 m l. of f orm icacid per liter) w as started into a m ix ing cham bercontaining 2 500 m l. of solution # 1, thus initiatingg rad ie nt e lu tio n. Py rim i din es c on sis tin g p rima rilyof 5-ribosy luracil and uracil w ere eluted f irst, f ollow ed by the v arious purines, w ith uric acid beingeluted last. T he com b ined ef fluents containing thepurines (ex cept for uric acid) f rom three suchf ractionations on D ow ex 2-f orm ate w ere ly ophi-liz ed, and am monium form ate w as rem oved bysu blim ation at 55 -60 . T he residue w as tak enup in 50 m l. of 0.01 N HC1, added to a colum n(46 X 2.5 cm .) of Dow ex 50-H+ (X 12, 200-400m esh) w hich w as in equilibrium w ith 0.01 N HC1.

ultrav iolet lam p (253.7 m ^i), and tw o spots f requently observ ed w ere characteriz ed by brightb lu e f lu ore sc en ce and d id not appe ar to c orre sp ondto p urin es p rev io usly rep orted in u rin e (2 4, 3 3, 3 4).T o obtain these substances in larger quan tity w ithsu ff icien t p urity f or id en tif icatio n p urp oses, f ractions f rom the large-scale procedure containingrelativ ely high co ncentratio ns of the com p oundsin question w ere streak ed along the base line off il te r p ap er and s ub je ct ed t o one -d ime ns io nal c hro -m ato graphy . T he bands correspon ding to the compo unds to b e isolated w ere cut f rom sev eral papersan d w ere concentrated by a technic prev iously describ ed (9 ). T he su bstan ces th us p urif ied w e re u sedf or d ete rm i nati on o f RF v alu es and e le ctro phore ticm o bility and w ere rechrom atographed and elutedf or d ete rm i natio n o f u ltrav io le t ab so rp tio n s pe ctraw ith a B eck m an DK -2 recording sp ectrophoto m -eter.T A B L E 1RFV A LUESFURINA RYOM POUNDNDTw o MET HY LA TEDUA NINES

V A LU ES (X 100) IX V A RIO US S OL V EN T S Y ST EM SCOMPOUNDCompound

i so late d f romrine-DimethyIamino-6-hydroxypurine6-Hydroxy-2-methylaminopurineFORMi-Bu505043HAcn-Bu686858HC1i-Pr373747FORMEtAc393929EtAcform440

T h e c omp os it io n o f th e v ari ou s s olv en ts is as f ol lo w s: FORM


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1826 Canc er R es ea rc h Vol. 23, December 1963th e s ame RF v alu es a s 2 -d ime th ylam in o-6 -h yd ro x-y pu rin e, se pa ra tio n from th e 2 -mon ometh yl-su b-stituted guanine on the sam e chrom atogram wasnot obvious. The color of the urinary com poundon chrom atogram s observed over an ultravioletlam p w as variable, depending on w hat C hrom atographie solvents were used and how soon thechrom atogram w as exam ined after rem oval fromthe solvent. It varied from a dark absorbing spotin ammoniacal and neutral solvents to an intensebright blu e fluorescent su bstance in th e isoprop yla lc oh ol: H 2O :HC1 so lv en t. W ith th e la tte r so lv en t

TABLE2ELECTROPHORE TICOBILITIESOF URINARYCOMPOUNDANDTwo METHYLATEDGUANINES

COMPOUNDCompound

i so la ted f romrine2-Dimethy lamino-6hydroxypurine6-Hydroxy-2-methylaminopurine0.02

MitratepH3.04. 5athodal4.5athodal3.8cathodal0.01

HBoratepH9.2000

MOBILITIES (CM.)*

* 4 ho urs, 8 00 vo lts , W ha tm an #1 filter p ap er.

of the leukemic urines with the small column ofDowex 2 resin, subsequent chromatography rev ea le d th e p re se nc e o f 2 -d im eth ylamin o-6 -h yd ro x-ypurine in the effluent adenine peak, and occasionally it also extended into the adjacent hypo-xanthine peak. Com parable aliquots of the effluentpeaks obtained from normal and leukemic urineswere used for paper chromatography, and onlyonce w as the dim ethyl-guanine derivative observedin the normals (seven cases), whereas it was easilydetected in two-thirds of the leukemic specimens(30 cases of a variety of types including acute andchronic granulocytic, lym phocytic, and m yelocyt-ic). The compound was routinely detected chro-matographically in the effluents from the Dowex50 columns used in the large-scale procedure w ithboth normal and leukemic urine; but it was notconfined to a single peak, and a quantitative estimation of its level has not as yet been undertaken.W ith a visual inspection of the chromatograms, arough classification of the concentration could bemade on the basis of spot size and intensity, andit appeared that the acute leukemias and chroniclymphocytic leukemias were associated with anincreased excretion of this compound in comparison with what was found in normals and chronicg ra nu lo cy tic le uk em ia s.

T AB LE 3ULTRAVIOLETBSORPTIONHARACTERIST ICSFAUTHENT IC-DiMETHYLAM iNO-6-HYDROXYPURINE AND THE COMPOUND ISOLATED FROM URINE

COMPOUND2-Dimethylaniino-6-hydroxvpurine

Compound i so la te d f rom u ri ne pH

l>w256,

285*255, 285*Xmn233234pH

li^roa245,

279*245 , 2 79*Xn ,i n2 6827 0* Inflection.

6-h ydroxy-2-m ethylam inopurine exhibite d co ns id era bly le ss flu ore sc en ce , a nd th e c olo r w as d arke r. In sid e-b y-sid e C hroma to gra ph ie c ompa riso nsof the isolated com pound, synthetic 2-dim ethyl-amino -6 -hyd roxypur in e, and 6 -hyd roxy -2 -me thyl -am inopurine in a variety of solvents, the isolatedc ompo un d ro utin ely e xh ib ite d th e same g ra da tio nsin color as th e authentic 2-d im ethyl-substitutedguanine.In Table 2 the electrophoretic m obilities of thecom pounds are given, and in Table 3 ultravioletabsorption spectra are presented. Elion, Lange,and Hitchings (13) and Sm ith and Dunn (29) havereported previously spectral characte ristics for 2 -dimethylamino-6-hydroxypurine.After fractionation of the purines in a num ber

T he ribosyl de rivative of 2-dim ethy lam ino-6-hydroxypurine was isolated from the urine of anorm al subject. It was noted on paper chrom atograms prepared from an effluent fraction of thepurine s obtaine d from c hrom atogra phy on D ow ex2-form ate resin, and a sufficient quantity w as isola te d b y p ap er C hroma to gra ph ie te ch nic s to p erm itidentification. Its Rp value in sec-butyl alcoholsaturated with w ater was 0.35, but it rem ained atthe o rigin w hen borate w as added (8). T he m arkedly lower RP value in the presence of borate wasconsistent with a ribonucleoside structure. TheC hrom atographie and electrophoretic prop erties(m igration toward the anode in 0.01 M boratebuffer, pH 9.2) of the urinary com pound agreedwith those reported by Smith and Dunn for 2-di--



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FINK et a l. U rina ry %-D ime thyl amino -6 -hyd roxypur in e 1827me thyl amino -6 -hydr oxypur in e r ibonucl eo si de , andthe u lt raviol et absor pt ion spec tr a a ls o cor re spondedto the ones shown by these investigators (29).R ib os e and 2 -d ime th ylam in o-6 -h yd ro xy pu rin ew ere detected on chrom atogram s after hydrolysisof the urinary compound in l N HC1 at 100C . for1 hour. The sugar component was visualized onc hroma to grams b y th e a nilin e h yd ro ge n p hth ala tereagent (25), and the R F v alues agreed w ith thosereported for ribose (14) ; the purine base obtainedb y h yd ro ly sis w as id en tifie d a s 2 -d im eth ylamin o-6-hydroxypurine on the basis of its absorptionspectra and also by direct C hrom atographie comparison with an authentic standard, with theso lv en ts liste d in T ab le 1 . A c are fu l id en tific atio nof the nucleoside has been m ade in urine from onlyone subject, but a blue fluorescent spot in thep ro pe r Chromat og ra ph ie p os itio n fo r th e compoundhas been observed in urine from a number ofpatients. DISCUSSIONThe list of m ethylated purines found as m inorc onstituents of ribonucleic acids has continued toexpand since the initial report of Littlefield andD unn (19). It now includes 1-m ethylguanine (2,2 9), 2 -d ime th ylam in o-6 -h yd ro xy pu rin e (2 9), 6 -h y-d ro xy -2 -me th ylam in opurin e (2 , 2 9), 7 -me th ylg ua -nine (12), 2-m ethyladenine (18, 19), 6-m ethyl-amin op urin e (2 , 1 8, 1 9), 6 -d im eth ylamin op urin e(18, 19), and 1-m ethyladenine (11, 12). Thesemethylated purines occur in the sRNA (10, 5)involved in the transfer of am ino acids, but theyare not incorporated directly into the RN A (30).The m ethyl groups originate from m ethionine (6,22, 23), and the methylation is carried out at theleve l of th e pre-form ed polynucleo tide structureby a soluble enzym e system , R NA m ethylase (15).Further purification of the system has demonstrated that the transm ethylation is perform ed byS -adenosylm ethionin e and that specific en zym esm ay b e in vo lv ed fo r th e p artic ula r b ase m eth yla te d(16, 17). The function of these m inor bases in thesRNA has not yet been determ ined, and evidenceby S tarr (31) indicates that the m ethylation is notessential for the amino acid acceptor role of thesRNA. The question as to a possible coding function of the methylated bases was raised by Cantoni et al. (7) in a recent investigation in which2-dim ethylam ino-6-hydro xypurine w as found tobe the only methylated base in the serine sRNAo f y ea st .It se em s p ro ba ble th at 2 -d im eth ylamin o-6 -h y-droxypurine, as w ell as som e of the urinary m ethylated purines observed by W eissm ann et al. (33) name ly , 7 -m eth ylg ua nin e, 1 -m eth ylg ua nin e, 6 -

hydroxy-2-methy laminopurine , 1 -methy lhypoxan-thine, and 8-hydroxy-7-methylguaninerepresentm etabolic end-products of the sRNA. All but thelast two are known components of sRNA, and itis not unlikely that 1-m ethylhypoxanthine m aybe form ed bio logically fro m 1-m ethyladen ine (orits corresponding nucleoside or nucleotide). If 7-me thylguan in e i s a p recur so r o f 8 -hydr oxy- 7-meth-ylguanine, an oxidase oth er than xanthine ox idasewould presum ably be required, since it has beendem onstra ted that 7-m ethy lguanine is refra ctoryto xanthine oxidase (34, 36).O ther mechanisms may be mentioned whichm ight contribute to the urinary level of the m ethylated purines, but available inform ation is notadequate to assess them critically. F irst, it is conceivable tha t in some abnorm al state s an excessivemethylation of nucleic acid might occur, w ith aresultant increase in the level of the m ethylatedp urin es e xc re te d. T he exc es siv e me th yla tio n m ig htbe accom plished by donors oth er than S -aden osylmethionine, since M agee and Farber (21) havereported m ethylation of liver R NA and D NA afterin tra pe ri to ne al a dm in is tra tio n o f a C14 -la be le d c arc in og en , d im eth yln itro samin e. S ec on d, m eth ylation of purines other than in nucleic acid linkagem ay occur. T he early high specific activity of urin ary 7 -m eth ylg ua nin e a nd 8 -h yd ro xy -7 -m eth yl-guanine obse rved after adm inistration of la beledg ly cin e to s om e in div id ua ls w ith g ou t, p oly cy th e-m ia vera, and m yeloid m etaplasia (37, 38) couldbe consistent w ith som e non-nucleic acid purinem ethylations. A xelrod and Daly (3) obtained extracts of rabbit tissues which could methylateadenine to 3-m ethyladenine, but the latter compound could not be detected in DNA or RNA, andit or the corresponding hypoxanthine derivativehas not been identified in urine. Rem y reporteda n S -a de no sy lm eth io nin e tra nsm eth yla se o f E . c olithat could N -m ethylate some unn atural synthetic2-am ino-substituted purines, but it exhibited noa pp re cia ble a ctiv ity towa rd g ua nin e (2 6,2 7). H owever, in a footnote to a later report concerning am ammalian system for S-m ethylation of 6-thio-substitu ted pu rines, he stated that E . coli extra ctsc on ta in in g a n a ctiv e 2 -amin op urin e tra nsm eth ylase system synthes ized 6-hydroxy-2-methy lamino-purine in the p resence of S -ade nosylm eth ionineand guanosine-8-C 14; thus, the nucleoside or nucleotide derivative may be the natural methylacceptor, rather than guanine (28). Third, an enhanced level of urinary m ethylated purines m ayre fle ct a lowe re d m eta bo lism o f th ese c ompo un ds,such as demethylation or cleavage of the ringstructure. T ow nsend and R obins (32 ) have recen tly demonstrated that methylation at position 7



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1848 Can ce r R es ea rc h Vol. 23, December 1963re nd ers g ua no sin e a nd x an th osin e v ery su sc ep tib leto ring opening between C-8 and N-9 under m ildconditions of temperature and pH. Thus, if 7-m eth ylguanosine m onop hosphate from the sRNAwere b io lo gic ally d eg ra ded to 7 -me th ylg uano sin e,it is possible that a portion of it m ight undergocleavage of the im idazole ring yielding a 5-for-my lam in opyrim id in e d er iv ativ e. D eme th yla tio n o f6 -m eth ylamin op urin e h as b ee n d emon stra te d w itha rat liver hom ogenate (100,000 X g supernatantfraction), which yielded hypoxanthine and uricacid as two of the products (27), but biologicaldem ethylation of the natura lly o ccurring m ethylated guanines has not been shown, and they areeither refractory to xanthine oxidase or acted onat an exceedingly slow rate (34, 36).The increased urinary excretion of som e of them ethylated purines in many of the leukemic patients is of considerable interest, but the signific an ce o f th at o bse rv atio n awa its fu rth er in ve stig atio n o f th e b io ch em ic al m ec ha nism s in vo lv ed .

ACKNOWLEDGMENTSW e gratefully acknow ledge our appreciation to D r. G ert ru de E li on f or a g if t o f 2 -d ime th yl am ino -6 -h yd ro xypu ri ne a ndto D r. J. D . Sm ith and D r. G eorge B . Brow n for sam ples of 6-hydroxy-2-methyIaminopurine.REFERENCES1 . ADAMS,W . S .; DAV IS ,F .; a nd NAKATANI,M . F urine andPyr im id in e Exc re ti on i n Norma l a nd L eu kemi c Sub je ct s.Am. J. M ed ., 2 8:72 6-3 4, 1 96 0.2 . ADLER,M .; WE IS SMANN , .; a nd GUTMAN ,A . B . O ccurrence of M ethylated P urine B ases in Y east R ibonucleicA ci d. J . B io l. C hem., 2 30 :7 17 -2 3, 1 95 8.

3. A XE LROD ,., and DALY, J. T he E nzym ic C onversion ofA denine to 3-M ethyladenine. B iochim . B iophys. A cta,6 1: 85 5- 56 , 1 962.4 . B ERGMANN ,F .; KWIET NY ,H .; L EV IN , G .; and E NG ELBE RG ,H . Studies on the E nzym ic O xidation of A mino-p ur in es . B io ch im . B io phys . Ac ta , 3 7: 43 3^11 , 1 960.5 . B ERGQUIST,. L ., an d MATHEWS ,R . E . F . O cc urre nc e an dD istribution of M ethylated P urines in the RN A of Sub-c el lu la r F ra cti on s. B io ch em . J ., 8 6: 30 5- 13 , 1 96 2.6 . B ISWAS ,B .A . ; EDMONDS, . ; a nd ABRAMS ,. T he Me th yl -ation of the P urines of Soluble Ribonucleic A cid w ithM ethyl-labeled M ethionine. B iochem . B iophys. Res.C omm ., 6 :1 46 -4 9, 1 96 1.7 . CANTON I,G . L .; R ICHARDS,H .; a nd TANAKA,K . A C od in gF un ctio n for th e M eth ylated B ase s in S -RNA ? F ed. P ro c.,2 2: 23 0, 1 963.8. C LIN E, R. E.; FIN K, R . M .; and FIN K, K . Synthesis of 5-S ubstituted P yrim idines via F orm aldehyde A ddition.J. Am. C hem . S oc ., 8 1:2 521 -2 7, 1 95 9.9 . DAVIS ,F .; DOBBS ,C . A .; an d ADAMS,W . S . E lution C oncen tratio n o f P ap er C hroma to gram S po ts . A nal. C hem .,3 4: 17 5- 76 , 1 962.1 0. DUNN, D . B . A dditio nal C om po nen ts in R ibo nu cleic A cido f Ra t- li ve r F ra ct io ns . B io ch im . B io phys . Ac ta , 3 4: 28 6- 88 ,1959.11. . The Occurrence of 1-Methyladenine in Ribonuc le ic Ac id . I bi d. , 4 6: 19 8- 200, 1 961.

12. . The Isolation of l-Methyladenylic Acid and 7-Me th yl gu anyl ic Ac id f rom Ribonuc le ic Ac id . B io ch em . J .,8 6: 14 P- 15 P, 1 96 3.13. ELION, G. B.; LANGE, W. H.; and HITCHINGS,G. H.S tudies on C ondensed P yrim idine S ystem s. X III. S om eAm ino -su bstitu te d D eriv ativ es o f G uan in e an d 6 -T hio -g ua nin e. J. Am . C hem. S oc., 78 :2 17-2 0, 1 95 6.14. FINK, K; CLINE, R. E.; and FINK, R. M . Paper Chroma t og ra ph y o f S ev era l C la ss es o f Comp ou nd s: C orr ela te dR F V alu es in a V arie ty o f S olv en t S ystem s. A na l. C hem.,3 5: 38 9- 98 , 1 963.15. F LE IS SN ER ,E ., and BOR EK ,E . A N ew E nzym e of R NAS ynthesis: R NA M ethylase. P roc. N ati. A cad. S ci., 48:1199 -1202 , 1962 .16. . Properties of the Enzyme System Which Methyl-ates S olu ble RNA . F ed . P roc ., 2 2:2 29 , 19 63 .1 7. GOLD,M .; HURWITZ, .; an d ANDERS,M . T he E nzymaticM ethylation of R NA and DNA . B iochem . B iophys. R es.C omm ., 1 1:1 07 -1 4, 1 96 3.1 8. L ITTLEFIELD ,. W ., a nd DNN ,. B . N atu ra l O cc ur re nc eo f T hymin e an d T hree M eth yla te d A de nin e B as es in S eve ra l R ib on uc le ic A ci ds . N at ur e, 1 81 :2 54 -5 5, 1 95 8.19. . T he Occurrence and Distribution of Thym ine andT hree M ethylated-adenine B ases in R ibonucleic A cidsf rom Sev er al S ou rc es . B io ch em . J ., 7 0: 64 2- 51 , 1 95 8.2 0. MA cN uTT, W . S . T he E nzyma tica lly C ataly se d T ra nsfero f t he D eo xy rib os yl G ro up f rom One Fur in e o r P yr im id in eto Ano th er . B io ch em . J ., 5 0:3 84 -9 6, 1 95 2.21. M AG EE, P. N ., and FA RB ER ,E. Toxic Liver Injury andCa rc in og en es is . Me th yl at io n o f Ra t L iv er Nucl ei c Ac id s b yD im eth yln itro sam in e in V in o. B io chem. J., 8 3:1 14 -2 4,1962.2 2. MANDEL ,L . R ., an d B ore k, E . V aria bility in the S tru ctu reo f R ib on ucleic A cid . B io ch em . B io ph ys . R es. C omm ., 4 :1 4- 18 , 1 961.23. . T he Source of the Methyl Group for the Thym ineo f RNA . Ib id., 6:1 38-4 0, 1 961 .24. P ARK, R . W .; H OL LAND ,F .; and JE NK IN S, A . U rinaryPur in es i n L eu kemi a. C an ce r R es ., 2 2: 46 9-7 7, 1 96 2.2 5. PARTR IDGE,. M . Anil in e Hyd ro ge n Pht ha la te a s a Spra y

in g R eag en t fo r C hroma to grap hy o f S ug ars. N atu re , 1 64 :4 43 , 1 949.26. REM Y, C. N. M etabolism of 2,6-Diaminopurine: S-Adenosylmethi on in e a s Me th yl Dono r f or 2 -Me th yl am in o-6 -a mino pu rin e S yn th esis. J. B io l. C hem ., 2 34 :1 48 5-91 ,1959.27. . M etabolism of 6-M ethylam inopurine: Synthesisa nd D emeth yl at io n b y E sc he ric hia . c ol i. I bid ., 2 3fc :2 99 9-3005 , 1961 .28. . M etabolism of Thiopyrimidines and Thiopurines.S -Me th yl at io n w it h S -Adenosylmethi on in e T ra nsmethy l-ase and C atabolism in M ammalian T issues. Ibid., 238:1078 -84 , 1963 .2 9. SMITH ,J . D ., a nd DNN ,. B . T he O cc ur re nc e o f Me th yla te d Gua ni ne s i n R ib on uc le ic A cid s f rom Sev er al S ou rc es .B io ch em . J ., 7 2:2 94 -3 01 , 1 95 9.3 0. S TARR ,J. L . S tud ies on th e M eth yla tio n o f S olu ble R ibonucleic A cid. I. F ailure of the D irect Incorporation of 6-Me th yl am inopu ri ne . B io ch im . B io phys . Ac ta , 6 1: 67 6- 80 ,1962.31. .h e In co rp oratio n o f Am in o A cids in to "M eth yl-p oo r" Amin o A cid T ra ns fer R ib on uc leic A cid . B io ch em .B io ph ys . R es . C omm ., 1 0: 18 1- 85 , 1 96 3.3 2. TOWNSEND ,L. B., and ROBIN S,R. K. Ring Cleavage ofP urin e N ucleo sid es to Y ield P oss ib le B iog en etic P re cu rs ors o f P terid in es a nd R ib oflav in . J. Am . C hem. S oc., 86 :242-43 , 1963 .



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FINK et a l. U rina ry -Dimethy lamino -6 -hyd roxypur in e 18293 3. WE ISSMANN,B .; B ROMBERG,. A .; and G TMAN ,. B.T he P urine B ase s o f H uman U rine . I. S ep ara tio n an d Id ent if ic ati on . J . B io l. C hem., 2 24 :4 07 -2 2, 1 95 7.34 . WE IS SMANN ,., an d GTMAN,. B . T he Id en tifica tio n o f6 -Succinoaminopu rine and o f 8 -Hydroxy -7 -methy lguan inesNorma l Human U ri na ry Con sti tu en ts . J . B io l. C hem.,2 29 :2 39 -5 0, 1 957.35 . WYATT,G . R . T he P urin e a nd P yrim id in e C om po sitio n o fD eo xy pe nto se N ucleic A cid s. B io ch em . J., 4 8:58 4-9 0,1951.

3 6. WYNGAARDEN,. B . 2,6 -D iam in op urin e a s S ubs tra te an dIn hib ito r o f X anth in e O xid as e. J. B io l. C hem., 2 24:4 53-6 1, 1 957.3 7. WYNGAARDEN,. B .; B LA IR ,A . E .; an d H ILLEY,L . O n th eM echanism of O verproduction of U ric A cid in P atientsw ith Prim ary G out. J. Clin. Invest., 37:579-90, 1958.3 8. Y tf , T . F .; WE ISSMANN ,.; SHARNEY , .; KUPFER ,S .; a ndGTMAN,. O n the B iosynthesis of U ric A cid from G ly-c in e-N1 5 in P rim ar y a nd S ec on da ry Pol yc yt hemi a. Am . J .Me d., 2 1: 90 1- 17 , 1 95 6.

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The Identification of 2-Dimethylamino-6-hydroxypurine and Its Ribonucleoside in Urine of Normal and Leukemic Subjects