m m & m m s m m u highek vims m animals st Kctoort L« Jtaaill A THESIS Submitted to the School of graduate Studies of Michigan State Daiooreltgr of Agriculture and Applied Science la partial fulfillment of the requirements for the degree of DOCfOK QT PHUOSOPM? Department of Chen&etiy 1955 ProQuest Number: 10008323 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008323 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 S/lin /oil fl» outlier wishes to sapress M o sincere appreciation to Dr. Diehard 8, Byerrwn for M e interest, guidance, md counsel, which greatly facilitated tiw of w * problems« Be also wishes to thank the other members of the Chemistry Department for their helpful counsel from tine to tine; and also special gratitude to Robert L* Berman for M e assistance and helpful suggestions. finally, tbs writer wishes to thank the itoolo Energy Commission and the Chemistry Depart* neat of Michigan State University for providing funds in support of this work. iHteeeaettm* iW W W * ” * • « # 5W St1JMJHIHffiftlfh BfaWWr JULStflLaa WlflfW oo * ii USA She wither nas hem March 13, 1927 in loungetoon, Ohio, end received hie secondary education at Woodrow Wilson High School in Teungsteua, He served as * laboratory technician In the United States Havy Medical Corps for tee years, and entered feengstem College in January 19h7# He transferred to Ohio University in September 19b8, and m* graduated in Jane of 1950 with a Bachelor of Science Degree, He enrolled in the Graduate School of Michigan State University in the fall of 195C as a feasting Assistant in Chemistry, remaining at that position until recalled to naval service in June of 1951, After completing a year and a half of duty as a biochemistry instructor, he resumed M s studios at Michigan State Gnlvsrslty in the fall of 1952 as a Special Graduate Research Assistant under an Atomic ftmrgf Gemnission Grant, Be received the Master of Science Degree in June of 1953, presenting as his thesis "The Hole of the AlphaCarbon of Glycine in Methylatlea Studies In Tobacco Hants,* Ms is married to the former Mevitta Floyd of Beaufort, S, C., and has a daughter, Sebette Aim, Upon graduation the anchor will be associated with Ml Lilly and Company as a research biochemist. ill mwmnMtm m m r m m gmma t u a w aid umiim mttMffv It* MNm Aa 4jt jBsnniiei %$ the ScIkwI #f Oradnate Sttidiaa #f W o M ^ State LPPv ®ratli iOTsfHWf 4sy^W5IWL*t»Pr^y a iwa&n**t fia-tamMi WWHWWtPw ^ ’ ■Pa fPd PHf WWWWfpP 1^tMn F of iT W»a »? ^P |j| |^f%||^t i!3ilS®|( Of Ht9 fWpdbPSlNlSBS: for $h# ^ g m of IHX?T^R Of ffiSIASNSQtWt' 33©jWMS*fclM9Qt Of OhondUHu^ W5S XM r iv 9t 8B8S8^I® 8$88Jfd(^§SSl8^ife888!^^fe938^P888^jjjj^3fe98®8!^® WW' I^IlfV In W Mj5&att** lja t^ mM wv^y^I^*4i1jm» pwmwwpf gtifettttMlttiSfll “ Mtw B»wfflj|WW'n^w W igjM ii^a QQm pA^iiJ ^vip W|((Pw» tkm^wtaUm*™ flrMMttdai4 XfcS'Jilb % B -fc awv W ( Mpy\ ”L ^ y^ pw^ i^ y afl# bat AllL.itr t jfiMMMfcAUffe 8fe-HtoifedMAA.ttA4kdfc.M(ft^‘'PfaMHik UliAili_A-ilfclA1 iM.jEL ifttk {^jg%~i~Mrjljrt'JL Yrfi^t9-Awfe.dAtA i jjp'WBp psmswxW^Pm mSar mmmmmm W t IB P iC w pcwEmiJ |wm«mm5bWP »*w0^ 9} 4 9S9l99a/$Mhm pjy# *k ¥• A# mm/mm ttimtiWyiKMPS AttF H.-'itf«tiai« ^P^Ifl^K-w ^Pw psRBP(w^pWWPw^wlwWp dpjT J^BSffwRBp^WnHj^sWP^ Jwdffi^F v@6 ^S^^iMPbvKw^l^m jP^*nP ^MpM^U^Mir IP* tffc tffw {i^fc pgpP^s -*4 te1| dfc M« til& dM ^4 M!&i4f ^pS^WKwHf l ll jffAi:^"4 fiP si6P $uS&S rM ow ifA w Iw Iffiw -<*a.%fr-jii^jAMjMhAfr. 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AFFISQIX t o 20ito«..##^ # 36 , e , 1*2 11SJP Of T1B1SS M B * 1 H HI Xf W ® CoBqxwltion of t o took Statute*S o l u t i o n , * , . , , . . , , . 7 t a m M m Of £«dl<*a«tiviiy fit t o toofetao Holocule Aftor t o t o i t o t o M o * of Hi CoopaitBoik of Various Coapouada as M e t o s y l Group t o s m a o * I * fto e o o »«»**»****«*•••««».*■*****.** 2® Incorporation of FotoidelsydiM5 lot© Eat EKA 0aflpaasttts«w»«#, S6 INTRODUCTION 1 wmmmm The investigation* undertaken here were concerned with some of the possible metabolic methylation reaction* in higher plants and animals, the first study was to examine compounds which night simply information about precursors for the H«methyl gnap of nicotine in tobacco plant metabolism, The second investigation s o a comparative study of the possible O-ewthyX group precursor* for the ligniis molecule in the tobacco plant. The third study dealt with the possible incorporation of formaldehyde into the C-methyl group of thymine and the ureide carbon* of the purinse. Transwethylation was first shown to be a reaction la animal metabolism hr du Ylgmeand in 19h0 (l). Sines that tins a number of compounds have been examined in animals and have been shown to give rise to methyl groups either by direct tr«j»metiylation or by reduction of a ^ooo^^esrbom unit to a siobhyl group, The origin of methyl groups in higher plants was not studied until later, although Barrensciieen and von Vilyi lagy in 19i*3 (2) found that the creatine content of wheat gem mas increased upon the e«*^fitatratton of glyoo— eyamiae. In 1902 Brawn and Byerram (3) showed that formate and the - methyl group of mothLonine could servo as B«methyl graup praoursers for the alkaloid, nicotine, in the tobacco plant, methionine being in­ corporated to a greater extent than formate into the methyl group, it about the sane time, Marina, Kirkwood, and eo-workers (U found that 2 methionine could eater the methyl groins of th® barley Alkaloid*, bordcntne, N-mebhyl iyrwata®, mad granting t© a greater extent than formate. They however noted tiiat the methyl group of choline did not give rise to the methyl group of hordeaiae., Byerms and $&»g ( 5)in studies using tehOBfio slants. observed that the mthtfi sreun of could aot as a precursor for the methyl grsqp of nicotine and was incorporated at about the same rate as methionine. That amtii serve as a emattrssr for the 0<4elkvl ***** SLMwtettvl eponns of 4tW> caster bean alkaloid, riclnlme.. was noted by Bubeek ***d Kirkwood (6). but it was also found that cholins and formate failed to give rise to these methyl groins, the reason postulated for the failure of clioline to serve as a methyl group precursor in the barley plant was the possibility of the absence of an eaeyne system to convert choline to betalas sines bstalne has boon shewn to be a methyl group precursor in aaimaLs (7) and recently in plants (@)« Direct transmethylation has bee® nheap to occur in higher p^s^te by Byerrum, Floketra, Bewey and Ball (9,10), who by the use of methionine doubly labeled with c«rbon~lli and deuterium dbsewwd the same deuterium to cashcw-lk ratio in the methyl group of nicotine and the methoxyl group ef llgnin from beth tObaeoo sad barley as was is the methyl group of wstidamims. It was also whom that p»Qt<«i»dw entered the methoxyl group of barley llgnin to a greater extent than formate (10). The «3pha cartoon of glycine* when studied in the animal, shewed little ability to serve as a methyl group precursor. However when studied in the tobacco 3 plant (H), it m* stem to bs a# good if not bettor than nethionine and obelise as a methyl group precursor of nicotine. The alpha carbon of glyeelate <12) also baa been ahems to be a methyl group procursor. Serine, glycine, and glaciate appear to be interrelated in plant metabolism (13)» and other studies (Xii,lS) hare item the interconversien of gTyftino gerino^ with the alpha carbonof beta carbon of serine, It then ms of interest the to ascertain whether serine was a methyl group preoureor in plants, Serine has bees shewn to set~ as a methyl »sreun anbaalsIs the wpwwwt study serine '■—1"rfRSlBw oroowtor »,TRr rr;w■ inWWIPWIlimr g u rerww pa^in* VR RWTtfpwW w p ' RRtRwrww labeled with casbos^lii in the beta therefore was *Aj4 The unusual incorporation of the alpha carbon of glycine into methyl groups In plant retaiMsiicr also led. to the possibility that the carboxyl carbon ef glycine might have a role in nicoMne synthesis* Previous studies (15) in the animal have indicated that the carbonyl carbon is oxidised to carbon dioxide and, the carbon dSmdbis is not reduced to fornate or any ether one-carboa unit. However, since in plant nsetabolism the carixoyl carbon nay react differently blmat in the animal, it was decided to feod glycine labeled Kith carbon~lh in the eaiboxyl carbon to tobacco plants, isolate the sloetins, and determine its radioactivity. Tbs testhyl group is unknown in animal metabolism but does occur in the higher pleat* As mentioned previously Byerrum, Flokstna, Dewey, h and tall (10) found that methionine and formate could give rise to the metlwucyl group «f barley ligrdn, whereas Msmk and Kirkwood (6) ateilarly showed that methionine gave rise to the 0*«©thyl group of rieinlne. The latter iavestlgatlon however failed to show that fornate sad choline gave rise to the ricinin® &*wtihyl group, Further studies ©a ligain isolated frow the barley plant (10) sad the tobacco plant have demonstrated the occurrence of between /»»**>«» and the nothea^l group* Previous to these studies little was known about the origin of the lignin methesyl group# although Klason (16) suggested that formaldehyde Right servo as a msthosyl group precursor. It therefore seemed of interest to investigate a snsnber of possible precursors to determine whether they could give rise to sethesyl greens to compare the- ©actant of iftonrDoratlon of these comneunda into the methssyl group* the present study dealt with the isolation of lignin from the tobaoeo plants to which had 'been adeiniatered DL~«erin©~ 3-044# fermaldshy&M?m , DL~mtldordne-siebhyMS*4, ^Lyeine-a-C*4, glycine-l-C*4, eholiasweethyl^3,4* formated14, glycine boiainemeiigrl*^*4# and gXyeolate^Z-G**, The biosynthesis of the methyl group of thymine has attracted interest is recent years and has led to investigations of possible precursors Is animals. Formate (18) has been shown to give rise to the methyl group of thymine and to the carbons f wad 8 of the purines. Methionine (18) has been demonstrated to servo as a thymine methyl group precursor, and a source of the itrslde carbons of the purines. Serine, m studied by Hwy» and Sprinton (IP), has also been shewn to $ be a precursor for tbe methyl group ef thymine, and for the ureide oarbens ef the pptsw, It he* been indicated thet the carbon* mi nitrogen «f glycine enter the purine iselscuXe (Ip), and the alpha eafben lute the thymine methyl group, Formaldehyde else hue been suggested te have * role in the thymine methyl group syntheses, but so data te confirm tide suggestion has appeared* Heceat mark in the ret by fterxmana* Falrlsy, and Byeirua (I*?) in which methionine end formate mire studied, indicated that format® m e need te only tmlee the extent ef methionine ae a source ef the Upline methyl group, bat te about ten times Use extent of methionine as a source ef the oroide carbons ef the purines* they else neted that the methyl group ef methionine appeared te enter the methyl group ef thymine te a greater extent in relation to the nurime orelde thaH g vaptaiculito OS ft l$9n*snb$&S&t Supporting SSbSFial for the ptfffltff iffaUh nggp8 transplanted after a period ef two to weeks, flit pi**»♦•* ttft grows it ^ greenhouse »«»n1 a >>»4 about 6 inches tat attained, usually after tent t fO day grates period* They note fed twlm « teak with a nutrient solution which tee composed of $*8 g# 0*<»©s>** 1 g, %S04* ?H»0# 1 g, £»«** in k liters of U p water* Budding and flooring were toted It some plants during the feeding period, but for tit meet part wereabsent. fo prepare the for hydroponic auiii^fiietnti. effl'of the radio* active eoapsuxid to be studied, tin plants wsr® moved frow the flats and freed of vennieulib© as cos$Utely as possible. They sere then rinsed carefully with tap eater to m o v e most of the remaining extraneous naterlal and to avoid dosage to the roots* the roots sere soaked in 0,1 percent solution of a detergent garaicide (Detergent germicide go* 106 nsaafaotared by %sadotts fitelesX Co,, %amdette, Michigan) for m hear, with occasional agitation, to redoes the anther of bacteria present. The roots m m then rinsed with distillod water and placed in 125 «&• Erleaweyer flasks oentaining 5© ml. ef an inorganic nutrient elution. The nutrient solution was a lt| dilution of te stock nutrient solution shown In Table X, All t e weights are of te anhydrous salts and only T 0« ?» grade M M i mere «te. fe prevent t e destruction of te edmlailsiened erfsMile ««■.© jz »i «# 1*1000 b®* eolation «f aureosycin mas ate added to each flask* f^ B L K 1 CGKPOSITIOH OF fHE STOCK SOTKIEST SCUJTIOJI *%ber Calete aIterate ©a(i©»)t t tew sf Al sM em mw aX Ival ra a^« 2s es4 t# nJ 8s 5S fi taTX l•f© siw vf^ l?l Xs vmw 3lw BA gs' Ferri® chloride FoOl* 106© «i# 1 1« SN Qj m4 8 td^ tv #km «t nt^ 2 mg* Magnesium snlfate MgS04 25© ng« teoate sulfate (HR^JjjSO^ 25© mg, tSFa ii rts h'B 11 t^ yH imB*^d&'ltotatt&miuaeeinmfk # v ^ wSwr$ «Tit9 pbmplmte 25© mg. ffko administration of ttet vsj^ou* a^iyj mftw^apfca^ jeetbyl gromp nraetirssre irt.il bo deserted later* Hi ife© eameriiseats more C43Rpi#d oat la a special fmse ted to avoid any health haaard t e radioactive material* i*gfr,fc<»g mas need in an. tbo ex^rlnentsl mark for 12 hours each day. fiw soanee of ligbt consisted of * 13© matt incandes­ cent M b «ad tmo id inch, 30 matt fluoroacaat tubes, placed teat lb laelaos te*e t e top of te leave* and bad a li$*t intensity of 200*25© foot candles at te top ®f te plants, 1 stroma of osgrge® mas paste through t e matrtet solution of each plant t e t e minutes tmioo a day to provide aeration t e t e root systems. Additional nutrient solution mas added «* steed to keep t e metes constant. Following t e period of administration of t e possible methyl group precursor®, t e plants m m moved fro® te flasks, te roots mere 8 rinsed wUh distilled water, ant! %%t» excess lifuid blotted fra■ the w t * with « chess* sloth. The plants were theft out up into nail pieces and iwwdiately dried under infrared lamps. the temperature of the plants m s left «t 80#C, for an beur near the end ef the drying period. The dried plant materiel was finely ground is a mortar, nixed with 20 percent ef its weight ef calcium hydroxide, and placed Into a Kjsldahl flask, the material sea steam distilled until the distillate no longer gave a precipitate with silicotuBtstic acid,IisSieail*ii that us mere oicotiae was coming over. The distillate, which was collected in % ml. of 6 I? hydrochlorie acid, was cmoetitmted j& m & m te a mall velum and the nicotine was purified by two successive aeeotropic distillations iste 2 ml. ©f 1 Jf Wd imm alMlifte medium, m described by Saith (20). the distillftte was eenoentroted te dryness under reduced pressure and nicotine hydrochloride crystallised eut. The salt was dissolved in wMieiMtiMtfi wiw a «ann amount ef water, and a saturated aethanellc solu** Mim of nietvAt* Amid was added in excess. Aft®p a half hour ef standing the nicotine dipicnate crystals which had formed, were filtered off, washed with methanol and roerystalAlsed front hot water, the malting point was 223-22h°C (22k°C literature (21)). 14 Hethylfttlon Study with Glycine «SU0 Savona! studd.es (3,5,31,12) in this laboratory have shewn that tobacco plants can absorb various organic compounds through the roots. An earlier study using glycine « M 14 (11) indicated that about 2 mg. of glycine could be afcsoxbed by the roots in a period of about four days. 9 J4 til® hydroponic admtnigtmtion ef the glyeim -1-C , under study in 4 k fc ill - ^ > 1 rfiyQilf -M* fr ^ fh^ m .fl Jft* ■*■- •BP (Xt v iw l 8^p8V33WWwv®^ lOQIROCl X#&3iDJL€* Mnlolgtratlon if, glycine »4«C** 14 * fm eapsrlmomtal feeding trial® nave ran mith glycine -1-C f using 50 tobacco plants In eaeb experiment, the plants wre propared as «*e described previously and 1.33 x 10 moles ef glycine with a radio­ activity of 1 x 10® counts per minute mas added to the nutrient solution in each flash, the molar quantity and radioactivity mas calculated to bo equal te compounds previously administered (3,5,llA2)* After the seven day administration period, the nicotine mas isolated as the diplcrate as described in the previous section. The nicotine diplcrate mas ground finely mitb a mortar and pestle, and 60 mg. of it mas plated on tared aluminum discs for counting. Results So radioactivity mas found in the nicotine diplcrate from either 44 trial after feeding glycine -1-6 f indicating that the carboxyl carbon of glycine does not enter into nicotine synthesis under the experimental conditions used. te Methylation Study mith Serine -3«C Uptake of DL - Serine As mith other possible methyl group precursors, it mas decided to food senna to the tobacco plants from a nutrient solution througa the * Obtained from Tracerlab, Inc., Boston, Maos. 10 This procedure would thou make It possible te compare the extent of incorporation lata nicotine and lignin ef the bata • carbon ef serins with ether methyl group precursors. However, before the administration af the radioactive serins, It w i necessary ta ascertain the absorption rate af serine, and ta dstamine whether serine was toxic la lav concern* trations, or whether microorganisms on tba raota destroy or change tha amine acid before absorption. Therefore 2 mg, a£ SI* * serine was addad to 10 flasks containing nutrient solution, Tobacco plants were placed is 6 flasks, and 6 root fragments 1 cm, long placed in each of 2 flasks, Tba last 2 flasks more used as uniraculated controls. After toe days, the plants and roots vers removed from the various flasks, and the solutions analysed for remaining serins by the ninhydrin method of Harding and Maclean (22), This method was considered adequate since it was shorn that plants growing in nutrient solution without added serine excreted nothing to produce color with nlnhydrin under the conditions used. The analyses of the solutions cos* tainlng the plants showed that after two days all of the serins had disappeared. The solutions containing the roots, when compared with the unlnocwlated controls, shewed no decrease in the concentration of serine, Those results indicate that serine stay be readily absorbed through the roots of tobacco plants from a nutrient solution and that no destruction of the serine by microorganisms occurred under the conditions of feeding. The ne»»texicity of HI - serine In the concentration used was evidenced by the normal growth of the plant stem, production of leaves, II and growth ef roots, The above findings agree with the results obtained by Ghosh and Barrie (23) whioh indicate that BL * serine can be utilised by tobacco plant* for growth and nitrogen metabolism when administered in a nutrient solution in the presence of ammonium ions, ta Administration of DL - Serine -3-C la order te duplicate the condition* ef previous methylatiea studies *e that valid comparison* ef the extent ef incorporation late methyl group* could be undo between serin© and the ether compounds, the DL e •ovine was administered in the same molar sjuantiiy and radioactivity as had boon dene previously. The BL - serine was fed to 2 groups of 30 tobaeee plants as described in the proceeding section ef Preparation of *e Plants. Saeh plant received 1,6 x 10 nolos of 0L • serins with a a radioactivity of 1 x 10 counts per minute as measured using a thin-ond window Ckigor-Miiller tube. After the seven day administration period, the nicotine was isolated as the dipierate a* described earlier. The diplcrate was finely ground in a mortar, plated on aluminum counting discs, and counted for radio­ activity, Damethrlatioa of Blcotino As will be indicated later the nicotine from serine - 3-C** fed plants was radioactive, and therefore it was desirable to determine whether or not the radioactivity was localised in the methyl carbon. The demethylation of the nicotine was don® by Brown1* (2k) modification * Obtained from the California Foundation for Biochemical Research, lee Angeles, California, 18 of Pregl*s method (25)9 and the methyl group isolated a* metbyltriethylammonium iodide, a white solid suitable for counting. Since tbs nicotine dipicrate ms tuite insoluble and unsuitable for demothylation, 200 »g, of nicotioe diplcrate ms dissolved in sodium hydroxide sad the nicotine mas aaeotropically distilled into 6 H hydrochloric acid* the distillate m s placed into the reaction flask of the demetbylation apparatus and concentrated to dryness under reduced pressure, the reaction flask m s then attached to the demethylation train and the following reagents added te the flask on the basis of 50 mg, ef nicotine* 1*5 mg* ef ammonium iodide, 2 drops of 5 percent gold chloride solution, and 1 ml* ©f 1*7,3 percent hydriodic add. The gas-msMiig bubbler contained 1*5 ml* of 5 percent sodium tdosulfate-oadmiuoa sulfate solution te remove fcydriodlc add and iodine, the delivery tube dipped boles tbs surface ef a 5 percent ethastolie solution ef trlethyladne in the receiving vessel, which ms ceded to about «?0°C in a methyl cellosolve-carbon dioxide bath, A constant stream ef nitrogen, introduced into the side arm ef the reaction flask, m s passed slowly through the entire apparatus during the domethylation process* The reaction flask m s embedded la a copper oxide bath and m s heated to 200*8 in 20*25 minutes* Then the temperature m s raised slowly to 358*6©*$ and hold there for 1*5 minutes. After the heat m s removed, the flask m s allowed to cool and m s flushed for 15 minutes with a faster stream of nitrogen, the receiver was than disconnected and the delivery tube was rinsed with absolute ethanol, The rinsings were placed Into the roeeiving flask, this flask was stoppered, the contents mixed, 13 and «H«wtd to stand overnight at room temperature, The following day, most of the ethanol was evaporated ever an Infrared lamp with a slew stream of air directed across the liquid surface« the loot of the ethanol and triethylamine more removed In a woman desiccator, fin notbyltrlethylajojoonium iodide remaining mas a white crystalline eanpoimd, flat quaternary iodide was dissolved In a small amount of ethanol and plated with a miereplpette m a tared aluminum disc, The ethanol was evaporated over an infrared lamp, and the disc reweighed te obtain the neight ef the compound te be counted* tow!*! the radioactivity ef the niootine isolated from serine *>C u fed tobacco plants is presented in fable XX* The results are expressed in counts per minute per millimole at "infinite thinness" * The column labeled "methyltriethylamBoaium iodide" shews that most of the radio­ activity of the nicotine after the administration of radioactive serine is located in the methyl carbon. The difference in radioactivity ef the nicotine in the two experiments was probably due te seasonal variations in growth and metabolism since the plants were raised at different times of the year. Seme incorporation ef serine into the rings of nicotine may be indicated by the fact that net all the radioactivity was recovered upon demethylatioa. a* TABLE n m m m or w m m m m ik the kicotirk w h w d i i A i m u s m m t m m m m s m m ~j-c iaxianan Specific Activity (Counts per minute per millimole) Methyltriethyl» Bloctine Mpicrate mmmixm iodide AHroraiBsmv ItMpK1 of Plante 1 30 i a * xo* 3.5 * xo* 2 m 1,8 X XO 2.X x XO* r A sample of the calculations is shoim in the appendix. ligaln Studies Source of Plant Material The tobacco plants used in this comparative study core of the stroio Klcettaaa rustics L, var. buaili*. The nicotine of the plants previously bad been moved by steam distillation |** the presence of calcium hydroxide, and the resulting residue dried* Several groups of plants bad been fed different carbon-lfc labolod compounds. The labeled compounds, fed in eguimelar quantities, and in equal radioactivity, *ero as follow i Aa Dl-«iethioaiiie-»ethyl«-C • ia (3), sodium fe?mate«C ($), glycine betaine^thyl-C*4 (8), KUeertne (3), cholius-methyl-C S4 glycine-a-C** (11), glycine*!^*4, calcium glycolate-2-014 (if), and fcroaldohyde-C*4 (36)* *s Tb® moles of each compound fed per tobacco plant was l.U x 10 and tbs a radioactivity sag 1*0 x 10 counts per minute. * 1 m indebted to S. A. Broun, R, L. Riagier, C, S, Sato, R. K. Wing, and I*. «J. Beeey for providing radioactive tobacco materials. 15 fen to fifteen grans of Hie driad tobacco plant residue nee treated by the netted suggested by MaeDeugall and Belong (26) to laolete a lev nitrogen containing Updn, This Method consisted of tm lg minute extractions vitli ether saturated eater to reneve nitrogen containing Materials, a 20 Minute extraction with g percent acetic aeid to reneve sens CMMbefeydrates, and finally tee 15 wiaate extractions with m ethanol* bensene (l»2) mixture to reneve Has fatty Materials* 4 Waring Blender nut muni to diseerse the ♦,*>**»<■>*«» elast residua in the solvents. and cheese eleth need a* a filter to renew the liquid portion. The •BNPTCP residue Obtained series of extractions * Wv FWt gWr W*e 1He rtthe M M l WW WWB» w^HNsolvent BW^ W'BB.iFFWWNBF» bnnffl is eeler. was broken an into fine nieces. eleeed in a larse beaker a, <^4 eoswred ^ ^wes F* OWS^^wn os, with .smbfbows a wee b® ev ,ifed bfoo (usually SO bil.) ^wob* si. ^f of fo Jnereeat iwFBiFww^e (v/v) sulfuric aeid, This suspension mas allowed to stand 18 hours at g*C to hydrolyse and prevent carbonisation of the carbohydrates. then the sue* pension was diluted to tees percent, mid boiled gently using glass beads as aati-toaap Materials for two hours to complete the hydrolysis. The velum was kept constant by the addition of distilled water, sad then filtersd on a sintered glass funnel under rodeoed pressure. Difficulty was ensousstered la the filtering process since the funnel rapidly became cleggs*. This trouble was avoided by aUswlag the lignln te settle, mid dtcmitiwg the supernote before filtration. The dark brown llgnin was washed thoroughly with distilled water, partially dried on the filter with section* and then transferred te a vacuun desiccator te complete the drying process. ll&ixt 16 mrnimmrn of Mania $&mm lignin isolated fvm plaa*9 fed all of the compound* listed abam mm radioactive, *• will b* indicated later, it m s of interest t« ascertain whether any radioactivity- was located in the nethe*# group. f*e maiiistudies It was necessary to cleave the nethyl group am obtain it in a suitable cenpouiid far eetmtiag. Flok»tra*» modification (it) af Phillips* netted (28) warn used to ipMi the aethyi groa^ trm the oxygen la yield methyl iodide, the methyl iodide was swept into an ©thaneXtd solution ©f tidLethylawin© te fern ««! fl©tMt iodide column indicate that the majority of the ©arbon-Xl* la the labeled oen* pound* entered the metlMnql group of ligrtin. Th® beta carbon of eerine weald therefore appear to be the boot CMsethyl group precursor studied thus far, with feraaldelg«le being second best* Methionine? (3), the alpha carbon of glycine (31), and bias alpha carbon of glyeelate (12) giro results similar to the 8«M©ttayl group of nicotine precursor studies, is stated above in the llgain results, format© appears to bo iacorporatod to a greater extent into the O^mothyl group than into the Jtowithyl group ohm compared to the other precursors, It is not known why such low recoveries of count* in the aethoxyl group m$ mmmterad in the choline and bctaia© studios, but these are being investigated at present. The carboxyl earbon of glycine probably goes to eaxfcon dioadde and random distribution is obtained since the percent radioactivity of the lignim recovered on demethylatien was similar te the recovery obtained after feeding bicarbonate (29). 20 The difference la count* observed trm * particular compound la twe different experlweBts may be due te seasonal variations la growth and metabolism. 9Utt& yrt as nwmm* ccmmxsor or ?«c®s m m mmwms m toaqoo u a r a ttMwwwwfuw <«, were detewined. 4 Tracerlab internal flee Gfelgtr counter me wmd te measure t e radioactivity. Criteria a# fhe teenategs^Me imstima ef each nitrogen base were detei»ii»d te be satisfaeterlly pare a® fudged bgr t e m t e ef xv^loaetivity te MbBorimmy, and from te retie ef abeoataoiee at tee selected naira length#. tealte t e radioactivity ef t e deijosyribenaaleio acid mmpmm te isolated 14 from rat# fed fsmmJUiahjteG ie tens in fable If. t e epeclfic activity t e eapresaed ae cotes per minute per microm®!©, t e calculation ef tech ie nb&m in te appendix, them reeulta indicate that fomalda- hide ie incorporated lute th^aino, adenine, wad guanine te a much larger extent ten into eyteslaa. If t e dilution value ie regarded a# a measure ef utilization, it ie indicated that about 3 moles ef formaldehyde enter t e puttee for each mole ef forsaaIdehyde teefc enter# thymine. 27 the low radioactivity ta the eytesiae weald seem to lend support te the assumption that west ef the radioactivity ef ■W^wQWL i?3BWe®K®5H» SriWiflBMw w KUm is located In the M GOCftXCMdPdu ti® flW® w major role in the mtkrfUMm of pyrimidines, the radioactivity ef the aitaaiiiMf juiit gnfl»4m> ig t&eugfcb te he Iwatfftfl in the 2 Bfl*1 0 oarhe**# es deMSWstreted by ether wrfeere using formate serin®, end Mould indi** eete that formaldehyde weald be e relatively major source of the ui*oldo oasbons^ 29 mm$sm t9 DISCUSSOT the result* obtain^I from Hie etudiae using serliaHMJ44 indicated that the beta carbon ef serins can be utilised ee a tearoe ef the methyl grwp ef ateetiiie, Bsmver, the results obtained treat the studies miug J4 glycine-l-C cam te indicate that tbs cartwayl eastern ef glycine wtm net 11ted ter sleetiae srctbaais under the fidediMtes need. The daaaetfayl* sties ef the Iff’ elatiiMl free tfr* eeriimHted trebffflee plants demonstrated tfa**- meet ef the radioactivity is tN* m e located la the aethyl group. Serin® pwiid.eiisly bed been ehem te- be a methyl group precereer lit jKw4.MMit.ii- tm t » M i mpasgnt work 1» tikfi flTSt 8Vid®*t*Se e f Itff« *» In t foo ww^wt-wMjww rmaetioiia m^w* slants• Weisabach. s£Lw»*irand iw^iaPsa^^pdPTSem^^aapms * e-^ow^niv^wiafcaa'O^^aip is wwiwfe h* ^*aep« c^i ww> *^ ^^^^awpw. dtoriaeee mw( ^pwv^wpmw (32) end Jcncsen end Hosber (33) shewed M the beta carbon ef serina eeuld be utilised by the m% for the synthesis ef tie obelise methyl group. Aractein end Neubergpr (3k) else haw 4me®sh*mted the utilisation ef the beta eatbea ef seriae for the eyaibeel* ef the methionine methyl group. In studio# by Amstela ( 35 ) , It m e found th a t only the 1» * w H m 1earner gave rtee te methyl group# la the rot. B^L-eerlne m e need ie tbie preset etedy ae it m e aeemd that beth isenens ef the aai*x> acid mre need in plant metabelian te yield methyl groups. the difference in the uce ef the t m iso®©*# has net been studied is p la n t metabolism ee that clarification ef tlxie point nuet malt farther Investigation. 3© frnmmm afasMm iawwliring w r liii plant predate have demonstrated th a t mm nethyl &m $* e f m rnm Sm (3#M©)« glycine betalne (8 ), and fitw lli* ( * ) , the easfeene e f tm m te (3,10), and formaldehyde (36), and the alpha earbos* a f glycine (11) and glysialai* ( if ) <*euld *«s*» as aatlgA greup preeuraera, the in s tig a tio n s o f nicotine f«*»aiie» ia the tebaeee i&aatjspeiride m excellent eg$ertunity te na!» a comparison e f the W le a i eenpettatla fed a te * the m olarity «** w»dl«MicMvity ware teapt e fa lie la iii# , the carbon a f fomimldefcyde aaana te be u tilis e d a t * m tftEvjfcfe'w ft ttfta iiH ftK a owi ean^igse1 aniiMnhiMeane^Haikine' 4 ^ tt m ^nnannsnaans' Tene nen^ttAdhiM ne' ^aMidheeL<^Mb Ahh^ewk f lta^^a^aiat^^a T^^pertp^nentp epessmpos sbasees sindied^. tin isndb <4hhn^> LjeL^fct&^eas ^nethe»kM. aaa^ tattar at ataaat free *Pr* lsfrwjiitfHt p lm ti fed 14 1 nan ebeat twice m iw&di©notlv© a t nl^ e tf mr fynm plants #nil iA *fa V j| ) | i| | j| m m a^ttr it dfc9tnw\ja$. f I* 4il it *fi ft t«U!l4«ltifci*^fe$wta Mi Jtft£ I$ N* w1 a9 i8 i1 ' Ja&w&w? «1an^i m &f *w *m A ^ m 3 «*wv.nt »$ l&o aep ' and Kieliuk «aei Bakasl (24) at the esidatloa «bat« ef fowsaldehyde appear to be attractive for the otUioatieji of formaldehyde, the alpha carbon of glycine, aai tfeo beta carbon of eeiim far tip formation of H*®etteyi group* in state* tfete "aetiirn mm&waA* qw M then bo reduced m tip «*»i»4ato!Ie» group* of to feist sethyl groups for llfeis* tt** and %rtr»*©B (1*5) denenetraM a lettered utilisation of •arise in. folio aetd deficloat rate to load ot^jpori to a feydrcjQ^thyl-* tstrahydrofolis add derivative of Kleliuk and Sakami. the addition of kcmocunitoixio W™'piWfw'TP^ ”T Pto fat wwt diets Wl^P thoe PWW been *faa**h to “©F of SBfiae (l?l «M«i foiftiat# into, *ananoowafi <>a ’ ■w^OPOFtIF tbs ©r, *wW*r < ■OaFOr *««* i»M$ jftpnaaa&aMi Jtt&tllvl ttraSOS to load siipperi to a hyvh<03iymsthyll^o8ieoyfitt©liKii dozioatiw * A possible pathway for the fbemtiift of &<»ebbyA gimps In the plant s^#it therefore be* Formate — ------- > *Actiee Foimto* **' (tains alpha m uw wW wk mm moNdwlbte t FoiMttafcyde----- >"ictif© Formaldehyde* t— -Sofia© beta Ktttl&t * group methyl group $b0 r©l© of tbe mnbosyl oafhen of glycine la plant metabolism appear* to be abobfc the same ao is animal mUbell*®. Sielan&i* and Greeifcarg (15) d©««i*trated that the carboxyl carbon of glycine m o 33 te eeztat dioxido and that toe eatoeft dioxide tamed k m not reduced to tamata* toereae the alpha carbon ef glycto* ***e rtoe te f«MKte# Waiaabaeh, £a«y» end %*toe@» (jt) else ehened that toe earbosyl cartoon ef glycine did aet 0 m rtoe te eheltoe notoyl group* M in the net* Xff the prweat oxpertoent aetog glycine**!*© had h e von fee * longer psriod possibly roatoa dtotribettoii of radioactivity la toe nicotine would m m been toned *« mx indicated by M.p {29) to atudio* with bicarbonate* Urnmmmm ft w i detMaeetrntad ‘ in fhewo tavoetti^tdUHi* of sethylabioii htosSjoe# toe ®f gty^tttt glytsolat®,, toe beta eostoee ef eorto®* toe eavthMi ef tomato awd toiaMtodehyd*$ a*** th® eetoaeyf etttoefi ef ah*ie eq ^® tj ah tSo^e AtaiM eaa aerve to iwtotoaent ef toe nagtoeayl earhon ef ligata* ^eeMeSe^i Mhti(aeaj jwk#e l e ^ de it4is iLaeo5 feaa< ki vinx hea d *ifca^a^* ^ ^otd (hi^^aMhfdv' ®fea*a l «H 9 r ^ ^ ivshm a g e w^ aeaat da radioactivity to toe lignin isolated tmm toe plant*, bet toe extent ef Amelioration touted eito toe *«b*t«»eea, After dentetoylation ef the lignie# it eee toned that meet ef toe vedteawtivlty ef toe lignin toe located to toe xnethoxyl >CW | groop ef ligato tmm plant* fed D^-esria®**, ^yetoe^dWS*4, tomal^toyd*-©1*, glyoolata-2-C14, and l m M U , the totoeayl gmeg* ef toe lignin after feeding efedtoe^toyl-C^ and h®taiii®-i»etoyl43U peeeeeeed only about «WMtot**t te #n##afto ef toe radtoactlvi% ef to® llgata, toereae the methexyl group of the ligalB trm plamb* fed #y8ine*l'«CW contained less than one-thirtieth of the radioactivity of the lignin. Tim ©-methyl group So unusual So the sense that it doos sot oxiot in talMl metabolism, ft m o first olioim by Byerrua and Vlokstra (k6) that tbo ©-methyl group of barley lignin could arise from the methyl carbon of methionine, and the carbon of fomnato. The nethieslas nothyl group one dononstrated to bo incorporated to a such greater extent than the formate. Dubeck and Kirkwood (6) found that the nothyl group ef motbionino could bo utilised as a source of the 0-methyl group ef rieinlae, an alkaloid from the castor bean, but they failod to show that either the choline nothyl groups or formate could give rise to the nothyl groups of this alkaloid, Sribnoy and Kirkwood (2*7) also have demonstrated that the nothyl group of methionine could serve as a precursor of the mstkylesediexy groups ef proteplno in Dlcentra hybrids, Subsequent studies by Byerrum, Flokstra, Dewey, and Sail (10) have shown that the methyl group of nsthieaime is transferred intact to give rise te the O-aethyl group ef lignin in barley and tobacco plants, an instance of transmethylation in higher plants, transmethylation in higher plants had been shewn earlier involving the methionine nothyl group and the nicotine methyl group (9), Studios by Stone CU8> in which labeled carbon dioxide was fed to wheat plants for a short time indicate that the methylation of lignin is an irreversible presses, the nothyl groups being of a «nem«4Sbilc* type. He demonstrated that the total radio* activity acquired by the ayrimgaldehyde portion ef lignin renalaed constant throughout the growth of the plant, indicating that lignin was 35 as end-product is the plant sad nethylatisn was a reaction is the syn­ thesis tf ligsis*, the results obtained is tbs present studies differ freo the result* obtained is previous plant nethylaties studies (3,5,8,11,12,36) mentioned is tl»« discussion ef striae Incorporation late the H-mothyl group of nicotine. The beta carbon of seriso appears to bo incorporated to the groatoot extent isto the 0-«*thyl groups of tobacco ligsis when compared with the other precursors* It is Incorporated to nearly twice the extost of the carbon of formaldehyde, about k tisos the extest of the alpha carbon of glycine and gLyeelate, asdi the methyl group of ssthlosiite, asd about 10 tises the extent of the carbon of formate, If only the L-serise is utilised by the plant them serine would be as ores better methyl group precursor who* compared te the ether eubstaaces* The reason for the low recovery ef the total radioactivity after desethylaties ef the llgsia from plants fed obelise asd betaise is set uaderstoed at presest and is under Investigation* The results obtained 14 fres the desethylaties of the ligsis from plants fed gLyciae*l«C seen to iadieate that the carboxyl carbon of glycine esters the ligsis mole­ cule readonly much as carbea dioxide esters the nicotine neleeule as demonstrated by Oulp (2?) • The inportast role which seems te be assigsed te the beta carbon of serine would Indicate that is higher pleats the 0*metfeyl group is formed at least partially by a different pathway than Is the H-aethyl group of nicotine, This suggests that possibly the beta carbon of serine is metabolised three#* more than one pathway* The possibility 36 of a "1-earben unit* (U3,hU similar te that proposed la the previous discussion seems to he attractive here also, bat sines the beta carbon of serine It laeerperated te a greater extent than the carbon tf formaldehyde there may be still another pathmsy. As mentioned previously, the mothylation ef lignin stems te be an Irreversible presses mhleb might lend support ts the idea ef the involvement ef the oxygen ef the serine hydroxyl grasp. Perhaps the serins condenses sith the ring structure ef lAgaia and then the carbon linkage between the alpha and beta carbon* is cleaved, followed by reduction ts yield the methexyl group. A Study using doubly labeled serine with 0 in the hydroxyl la group and 0 in the beta position mould be useful la proving this hypothesis. These studies also Indicate that the carbons ef formaldehyde and formate, and the alpha carbons ef glycine and glycolato might be utilised for methyl group synthesis by may ef the beta carbon ef serine as suggested in animal metabolism (Ui,3T). this again differs from the results obtained frsa the methylatien studies of the K-netfayl group ef nicotine. These investigations shamed that the carbon of formaldehyde and the alpha carbon of glycine mere incorporated to a greater extent than mas the beta carbon ef serine, mhleh suggest that they mere net metabolised by may ef serine. Incorporation of Formaldehyde into Thjmlne and hrrlnes The present study has shemn that the carbon ef formaldehyde can serve as a precursor of thymine and the purines, adenine and guanine. 37 t o lew toleactlvlty found in cytosine lends support to the assumption that all the radioactivity of thymine m o located is the nothyl group. Studies owing fo*wnte-CU , seariUis-.J-C*4, and glyelne~2-C**(17,19) have demonstrated ttit upon degradation of thymine newt ef the radioactivity was located in the methyl group, and in each ease the thysdso ring Isotope level eerrespeadod to the Isotope level of t o cytosine ring. It has been wan toon from investigations with pigeons that formate or biological precursors ef formate such as t o beta carbon of tevim, t o alpha carbon of glycine, and t o methyl group of choline could give rise to t o 2 and 8 carbons of uric add. Formate has boon shewn by totter, Velkln, and Carter (17) te give rise to t o methyl group of thymine in t o chick and rat* the methyl group of methionine formerly was found to servo as a rather ineffective thymine precursor by ®rown (18), but recently it was found te be readily converted to to 2 and 8 carbons of purines by Sfcae and Johnson (13) * J&wyn and Sprlason (I?) have shown recently that to beta carbon of serins t o t o alpha carbon of glycine could servo as precursors of to methyl group of thymine, demonstrating tot serine was incorporated to a greater extent than was gircto* The beta carbon of serine also was shewn to enter t o 2 and 8 carbons of to purines* Recent work reported by Hewnaan, Fairley, and Byewaa (1*9) dealt with a comparison of to carbon of formate t o t o methyl group of methionine as precursors of t o methyl group of thymine t o t o ureide carbons of t o purine* Earlier studies using formats (17) t o mstfciewlJie (18) had failed to provide a comparison of t o respective 30 utilisations for these precesses* Barcwura gj al. (2*9 ) found that fornate *a* « ness effective procurelor of the tlgmlns methyl group and the "mid* carbons of adenine and guantns than *ns the aethyl gnnp of weihioniae* However shea a ratio of the radioactivity of adenine to the radioactivity of thymine was considered, studios using formate p m ft ratio of about 5 to 1, whereas studies using methionine gars ft ratio of about l«fe to 1* These ratios indicate %too Methyl group of raothiosiao would outer the nsthyl group of thyslne acre preferentially than dees fornate wfcaa compared to the entrance into the oroide carbons of the purines, the work of Shg» osd Sprtssen (19) p m • oerrosposdiug ratio of 1*5 to 1 for the beta oarfoou of serine indicating that the methyl group of the beta oarber of eerie® may fern ft censes intormedlste before giving; rise to the welde carbons of the p H w and the aothyl group of thysin®, The present study using fernaJdehydo gave an adenine to tb^nino activity ratio of about 3 to 1, th e d ilu tio n v a lu e , th a t Is the s p e c ific a c tiv ity o f th e p recu rso r/ s p e o ifio a c tiv ity o f the oonpound is o la te d , Obtainod In the present study was eesspsred to the d ilu tio n values obtained fre a the format® and inethl&aiB® study Ch9) * They in d icated th a t fom aldehyde is ineorpoxmted in to the purines to about the sane exten t as fo rn a te , and about 9 tin e s the e x ten t o f the m ethyl group o f a o th ien la s , w ith respect t© Eowever when compared the incorporation in to tJ ^ a ln e , formaldehyde was used to tw ice th e e x ten t o f form at®, and k tin e s the e x te n t ©f m ethionine, those re s u lts seen to in d ic a te th a t formaldehyde has «s In ta r - mediate ro le as a thymine precursor when c e lla re d to aorta®, n s th lo n in e . 39 and formate, SLm^xk and (19) demonetrated that during the conversion of tl» Win carbon of eerine to th© thymine methyl group th© hydrogen* ©a the beta carbon ooeeapaay th© carbon. Whore** In th» eon* version to the wreide carbon* of th* purines extensive labilliatiea of th* hydrogen* took place, indicatlng too diotinot pathway* for tho too processes. In studies of th* synthesis of serins by Xisliuk oad Safcaal CftU) and by Kitano and Greenberg (39) , it m s suggested that an active internediat* confound, possibly a tstrahydrefolic acid derivative, at tho oxidation ctate of formaldehyde existed which could giro rise to serin© or t# the nsthyl g r o p of thymine and choline. Borg (h3) alee has proposed that hydroxythylhoBsocystoliie m o an important “active 1-earbcn intemodiato8» an* g^tapni (Ith) poatolatod an "active l-carboo eotopouiKi" at the oxidation atato of format* which might form the wreido carbons of th* purinee ©r bo reduced to fora tho "active ^rejEymathyltotrahydrofolic acid1* at tho oxidation atato of forraaldehydle„ These postulations would holp to explain tho difference in tho incorpora­ tion of formaldehyde and format* into tho nathyl group of thymine, Koceatly Greenberg (50) al3© ha« presented a aehono for th* interrela­ tionship of formaldehyde, tho beta carbon of serine, formate, and the purine oroide carbons utilising th* “active l-carben compound", a derivative of tetrahydrofolic add a* prepoood by Kinliuk and Sakarai (hh) , Wyatt and Cohan (51) have reported the occurrence of S^ydroajynotbylcytosine in phage d*©*jmbeaueieic add and proposed a pooaible pathway for the formation of thymine by way of the 5-hydroxymthylcytocina which secas to bo attractive in li#t ef the results with formaldehyde. mmm iiO sniffy X. The adaistisirattoii *f te tobacco plant* resulted la th® foxaatiw* «f radioactive nicotine. Hast of tho radioactivity was show* to bo cantered la the methyl group. 2* Whoa compared with other nethyl group precursors previously fed to tobacco plants, the beta-carbon of serins oeena to bo incorporated late the alootiae molecule la a looser anoimt than formaldehyde and the eXpti^corbon of glycia©, bat at about the sane extent oo yaothf©nine, ohellne, betslns, end glyoel&te. Possible tochodoo for tho fernation of the IMmsthyX group ore discussed* 3* After the administration of ipyoSaia-44!14 t* tOheooo plants, the alootiae dipierate isolated possessed ao radioactivity. It Is therefore that the eaiboQd OQ'rbfOt of gftyejm* does not enter late nicotine synthesis under the eoaditieas used, At h, ligota looted £wm tobooeo pleats fed a®rlae~3-C , formaldehydeIA AA 10 10'' 0 , «ethloaiR®-eiethyl«-C , $yei»sMM3 , glyciBe»l-C , choline* 10 SO 10 s* nethyl-0 , glyeiss botaine^matbyl-C , gJye©l*te-2«C , aad formate-C hts been shewn to possess radioactivity. Most If set ell of the radioactivity oss shewn to bo locetod la tho netheayl group except lo eftar feeding choline* betaias, end glyeine~l-C t Tho results with choline ead boteiao feeding ore unexplained at present, and tlie carboxyl carbon of glycine appears to result in rando® distribution of tho carbon In the lignla woleeule. $ A comparison of results indicates that the beta carbon ©f eerine la th® best aethoxyl cartoon precursor studied and formaldehyde is second best. Methionine, the alpha carbons of glycine aad glycolate are incorporated at about the same extent. Formate appears to be incorporated better into O-methyl groups than into N«as©thyl groups item cesapared to the other methyl group precursors. tka significance ©1 the results obtained are discussed in view o£ possible pathway®, 6, Eadieactlv© f©smldehyde, when injected iuiraperitoneally late rats, has been shoes te yield radioactive purines and pyrimidines Isolated from deoxyribonucleic odd. fhs purines* adenine and guanine* possessed a large amount of activity, as did th* pyrimidine, thymine, but cyteslme showed little activity. Host of th* radioactivity of thymine appears to be located in the methyl group. Iftiea compared with methionine and formate* formaldehyde appears te be intermediate la the formation of the thymine methyl group and purine urelde carbons* but seems te be a good source for both of these carbons. wsrmmm h2 BSPSRSWCEB *• I, njwM. 0kM., l - **1 " * SI, 9? (191*3). 3. Brown, S. A*, and Rywron, R. 4. ^ Chew, See,, 1523 (1952). It. Kirkwood, S., and Marten, t., Cm. 4, Chen., g , 30 (1951)* Hatchett, T# J(| Harden, t.# and Kirkwood, S., Can. 4, Chon. Jg., 1*88 (1953). UeU, I „ and Marlon, I*, Can, 4. Che*,, jfc, 61*6 (195h). 5. Byemiw, B. 6. Dubeck, and Hiag, B. E„ 4, Stol, Cham., JOg, 632 (1953)* and Kirkwood, S., 4, »!*, Chew., ^gg, 3«7 (1952). T. Manta, 4. A., 4. Biol. Chaw., 3J|, 1*69 (195»)* Wllliawa, 4. M*# 4r., Free. See. %»tl. Biel, Mad,, ||, 202 (1951), 8. Sate, 0, S., "Methyl Group Synthesi* In Plant H^taboliara'* B». 3D, Theala, Michigan State Bnlrereity, 1955* Spibaey, K., and Kirkneed, S., Can. 4, Cheat., Jf, 918 (195h). 9, Bewey, 1. 4«, Byerru», R. 26, 399? (19&), and Ball, C. B „ 4* Aa, Chaw, See., 10, Ryerron, B. 8», Floketra, 4. B*, Beway,1, 4,,and Ball, 4. Biel. Chen,, j&Q, 633 &95U • 11. Byarron, R, BiwiU, ». 1., and Ball,C. B „ at* 6hi (i9^t>» 0, B,, 4. Biel. Che®,, 12, Dewey, !». 4„ "Studies on the Bieayatfcesi* af Kicotlae and Ugsdn," Ph. B. Thesis, Miefcigaa State Bniwoity, 195h. 13. Tolbert, if , S „ and Cohan, M. S., 4, Bid, Chaw,, 2C&, 6k9 (1953). to ik, m ma, w„, a. bi«x. oh«., ij6, 9* (wto). m 1S* m *, VfW w ., j . w u a , a*,., m , f* *•» s w * * U . KIm q h , P „ Aridv. Keel* Rimma Geel,# £* Be. 13, 21 <191?}. I7# SSrebSi)1^ E#> m *"*"• °* s*» ** “ * • li, *♦ e« R»taitt# it, H#l aat Danuta* A* » „ 3* Biel, Che®,, 321, I t { it!4 ) , 18, Bwwaa* a, B„, "Phosphome MetabeAie®/ ISA. WL, HeSlrey, ». 0. and Giaea, B., e#s,f del® Hopkins Praee, Baltimore, i*t„ 1932, P* 38?, Siae, J* T., «ad Johnson, B. 0., t m » He, ht, Division of 8ie* “ "sal ‘C “hsroiii*tty, . ' 'See - Oity —- ~ ^‘patH‘ f.a^w Cbea&eai Comical See* Seeiety, Terk Heating, 193k* It. Sepe, B „ and Sprinscn, fi« » „ tf. Biel* Chaw., |g£, M>? (lt&h 2 0 . a«ith, c, r „ j m . sag, et«*., 2& (wto). 21, Brapy, I. * „ *n» Plairt t U a M * / fb* Blaldaton Co., Philadelphia, m s , P. 37. 22, Harding, ?, <1* and Kae&ean, R, Biel, Ohm*, Jg* a? (1913), 23. Okeefc, B, P., and 8b m A», ft* 1M Sadi Sc„ ][£, 187 <1930}. 2k* Breve, S* A*. "Studies m Reactions in Plantst the Origin ef the Hothyl Caafbea ef Rleoiin© Foreed by toeMaaft £EMSE»# Pb, s* fheeie, Richly state Ba&veMty, 1 » . 23* Frs#, F „ "QuaaUUiiv® Organl* meroanalyais," ktb 2%, Ed®., IhaBlaklitM S.„ Wltfditti, ISto, pp. «6. 26, HmOwcOI, S., w d Dalaag, W. A., Cm, 3. Kateareb, jja, kST, M 8 0*8). 27, neleetm, 4, ff„ "Peeaibl® Otigim of th® Hetheiyl Carbon ef Uguia Fernd by Hordern mOmmt*9 Ph. &• ffeeeie, HieMgen State Balirerelty, 1932* lib j, i n w , ofartud Agr, a**., |g, u s (1932). as. m u t p a , *?• 2?31®# ®« *•# ***• Bww»«i*tt9B ®f Bltdrtmwte late th® Hieotlno of M a a a safias* *. s. ***», m m m ««*• o*i.»wHjr, wsu. 36, CWm» W, s., jr. *». Q h w . S m . , 2 J, Cola, M. s ., Soloaoo, Jgg, lbn (wso). 377 (I9 li9 ). U . awnmrtm, S., Aat* Hod, Soand,, Bawd. jtf6 . 631* (19 M). ** £»£':#» Bj> “ * V 4 B “ B' “•* ' * te* ch“ - 8 53. 4mmmm, $* m& VMmv$ w, A*, J* Aw. Cta* Sac*, jj, 3316 teS©). 3fe. Ax*8tein, H* R* ? „ and tafcarger, A*, Bimtem* A*, j £ , 2$9 ( W l ) * 35. Aw8t*d®, I * R* T0 M 3^, m I mk , A.» !§ ,. 2? (U$L>» R i^gler, R# !»*, m d Hsa&H, R* I**, T e & m & i m Prae*» 3?# 3fc?fcol, A, A ,* ¥®Sjw , S ., M tfeu P*,# a m StoJUWs, K ,. BAol* Qmm,* I). |B * du VR fpm ft. ? *, fa r iy , W* 0 *, «$£ W||®a©, A, 1 ,, A* Aw* Otost, Sae„ a u *** IB* mtosa, 0*, msd M u f f B* H „ BAal, Own,, 396* $99 (3952). It©* Avmtmi®, H* R, ? *, «3*i Smsterg©**, ft*, Biaefeaai* A*, |g , 279 (1951). 10., A, A *, HNUwt I * * « tf 8Wt*»# S ., A, BA«3. mam*, J |g , 271 (1950). kt* & & * , ©*, %iMib«8b# A., «a* U , $09 tel). ill* * « « , P., 4. M * X . ©has., 2 ©* B „ J* A», ©Raw* See*, £, lii5 (1 9 0 ). W*. EUfliak, B. t„ «* Srtfd. w., J. An. Shorn, So.., Jo, lli56 (WSUj J, Biol. Chon. Sit, 1*7 was). US. Bins®, 6., «*) Sprlnson, B, B., J. Bl«l. Chesa., |Sj, M 5 (1950). iiS, B jx m n , B. 0., and fte h M m , J. H„ Federation Pro.., Ji, 193 (19®). 17* SribBsy, Um$ wad Kirteweod, S., R*tore, 171. 931 (1953)• 18* $ U m , I, 1## Can. 4. Ohem,, 20? <1953), 19* Honwai, &» t.f Fairley, J, 1#, aad Byemea, R* $*» Sec*, ££, 1902 W*$>* Clsam, 90* Greenberg, 0* B%, Federation Pres,, JJ, 715 (1951) • 51« Wyatt, G, S(| sad Ceheii, 3* S„ Biechom. 4., Jg, 171 (1953)* mwmu 1*6 m th* m u t f orrcuXa need in eerreot&Bg ike ebeerved count of aieoiiw diplewiit raethyitri«t^ri*B»»#ida» iodide to w t * ample thlefeaeee *n»* d » » im * m u d m epecific activity (cetmtey^diiute^llliaol#) Cm jfm * Gbaarv«d counts {ccunta/ainute) ff w ’wbftnw B K 00 e^9 w w O M v K V ' OfctO OBSHv^^ee' OWlinHliRli'inilV ^^v^eiBOtSnaS'HS es^e* OFw* sjB^spwwnsUpUn^E^wo W * neight ef sample counted . • fmotion ef mMrtiwBp* activity at ths fpewyle thickness need es >s$8ipjLe caic«ia«^wRi *syB3ti®B C„ * 112 B.fjs./al.. of Mnplo, A,, - 7 x 10s {2&tau, pH 8,25) A, - 0.257 3 ■^ilJS4^il2S2 *157 s i s loo - 3050 a.pj»./aM