carbon metabolism of c14-labeled aminoacids …carbon metabolism of c14-labeled aminoacidsin...

Carbon Metabolism of C14-Labeled Amino Acids in Wheat Leaves.II. Serine & its Role in Glycine Metabolism 1, 2

Dalton Wang &Department of Biochemistry,

\\ang aii \NVaygoo(l (24 in vestigate(l the coin-version of gl cine to sugalrs in xw heat leaves aindproposed a glyoxylate-serine pathwNN-ay in wN-hich serineoccupies a key positionl as anI essential interme(liate.Rabson et al. (19) also have (liscusse(l the glycolatepathway for the production of precursors of lhexoses.They hlave reviewed miiuch of the literature onI themetab)olism I) l)lants of gl--colic acid, glxovylic acidl,glyceric acid, glvcine. serine. a(ld relate(l interme(liates.

Glvcolic acidl is anI early p)ro(luct of photosynthesis(1), anId( a suhstantial l)ercenltage of the total carbonof the photosynthetic cycle elnters ilnto glvcollate(1, 25). This was shown dIrailiatically when Zelitch(27) blocked glycolate oxi(lation wi th an a-hvdroxv-sulfon<ate adI (lenmonstrated that approxi mately halfof the C14 fixedl appeared in glycolic aci(d. Kearnleyand Tolhert ( 11 ) shxow\edl thaLt Cl 4-l_Lhelecl glvcolatean(I phosphoglycolate appeared rapi(dly outsidle iso-

latedl chloroplasts photosynthesizing C 4,, aln(d theysuggestedl that these copopound(s likely are of splecialimportance in the tri-ansport of l)hotosvlnthate hetxweenchloroplasts an1(d cytol)lasm.

Glycine arises frolmi gl-oxvlate an(l in tnrln servesas a precursor fogr sel-inie. Rabson et al. ( 19 ) alsohave imlplicatedl glycerate aIS an intermediate hetxweenserine an(l hexoses, and have suggested that thisglycerate formiie(d hv the glycolate pathwaav representsa precnrsor for lhexoses inldepeln(lent of the phospho-glyceric acidl formiie(d from tlhe carhoxylaticin of rihU-lose (liphosphate.

In the present wx ork. an attempt has heen ivIa(leto sul)stantiate the role of serinle in the metaholisillof glvcine as wxell as to investigate the glvoxvlate-serine pathwxay in general. Afetabolic changes xxithtime in a numbher of laheled substrates have heenfolloxved in an attempt to clarify the interrelation-ships hetxxeen these simple organic acids and aminoaci(ls andl tlheir roles las precursors for suigar synthesis.

Received I)ec. 4, 1962.2 Publislhe( xith the approval of the Director of the

WVisconisin Agricultural Experinient Stationi. This inl-vestigation was supIx)rte(l in part by research granitsfrom the National Scienice IFoun(lation anid from theDivision of Researclh Grants, National Inistitutes ofHealth. This work x as presente(l at a meeting of theAmericani Society of Planit Phvsiologists, Oregoni StateI niv-ersity, Augu.st, 1902.

R. H. BurrisUniversity of Wisconsin

Materials & Methods

Plant Matcrial. See(llinigs of Khapli or othlervarieties of xv-leat xvere groxvn to the ealrly twxo-leafstage (ahout seven days ) in a Iplant groxvth chamherat 24' vith dlailv illumination for 13 lhonrrs at ahout2.000 ft-c. Sixty primary leaxves x ere cult for eachtreatmiienit, and tlleir (lx xxeighlt ran-ged firom (1.5 to0.7 g.

Fcdhing of C' 4-labec1d Substrate. The fee(dinigand tlle alcohol extraction of leaves and the snb-sequeint prelimlinary fractionation of the extraict intoaci(lic, hasic, and neutral fractirons xv-ere lerfcrme(lin the imianniier (lescribe(l previously ( 24

Serilne-1-C14 (0.1 Ilmc'2 IIlg ) and seriine-3-C1 I

l0.1 mc 2.6 ilig ) xere ohtained ftrolim Nexx EnglandNuclear Corp.. and thieir purities x-ere checke(d bvpaper ch romatographv with thiree solx elit svstens:a ) hutainol: Iaceticaci(l: vater (4 1:1 5 1)vphenol xwater (80: 20 xv v );: and c) ethyl ethieriforimiic acidl: xater ( 5: 2: 1 v x ). A samllle of ..'!cine-2-C'- (0.1 mic 5.68 migi) obtained fltromll lsot,(P)esSpecialties Co., xwats l)tIrified( before uise b l)?IlV)'chromatograpiy on W\hatman No. 3 i\1i1 l)ipaper xithi (le-elopiing Solvent of hutanol: (icetic acid : xxwater(4: 1: 5 v x ). (GlVcolate 2-C' xitl a sl)ecific alc-tivity of 0.041 mc mmole w-as ohtained fromil VolkRadiochiemlical Co. it xxsi usedl xithout fnr-thier pulti-ficatioin, althioughi it con1taLiinecla slighit iml)urity.

Ion-cxchangc Chr-omatographY of A 1)1mo0 A cidsa1(1 Organlic 4ci(ds. For the sellaration of individualamilino acidls usually the equivalent of 1 to 2 ng ofprotein hvdrolvsate xxas added to a 9 mm (liamiietei-analytical coltiinin of a Beckman amino acid anal r7ehaving an overall flox rate of 60 ml lhonir. The(livider xvas set to fnrni.,hli 1 @ho1-ur for- anilysis xx ithininhvdrin and( to pump the remainder to a fractiocollector xvhere 2.5 ml fractions xwere accumulated.Ani aliq(uot of 0.1 ml frolmi eachi fraction xx (a5 ;lkeifor the (letermination of ra(lioactivitv. 1'efereliceto figures 1 to 4 indicates thatt the amini) acids re-coveredl wxere highly radioactixve, single tuhes f;roli1the collector at times,e carrying as imluchi as 500,0)0)

Fractionls conlstitutinlg each amino aci(l pleak xx ercplooled. Recovery of the smllall (quanitities of amlinoctcidIs frolmi the p)ooledl solutionsxvas coml)licate(l Vthe lpr-esence of a relatively large quantltitv (t citrate

430

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WNrANG & BURRIS-AIETABOLISM OF C14-SERINE & GLYCINE IN W HEAT

from the eluting buffer solution. This difficulty was

overconme by passing each solution through a smallcolunmn of Dowex-1-X-10, chloride form, which re-

tainedl the citric acid and passed the amino acid. Theeffluent, containing the amino acid and a relativelylarge quantity of NaCl, was evaporated to dryness.The anmino acid was redissolved in a solution of ace-

tone ancl wvater (approximately 3 ml of acetone and2-3 drops of water; the concentration of acetone

should be such that the solution appears cloudy).An anmino acid recovered in this way as almostfree of salt. A recovery of 90 % or better could beachieved wvith three successive extractions. It shouldbe noted that serine recovered in this way gave twospots; one moved rapidly and one slowly on chro-matograms developed with ether: formic acid: water

(5: 2: 1 v,/v). The slower moving component con-

stituted about 80 % of the total. This change of Rfvalue also was observed with an authentic sample ofserine which hacl been treated in a similar way,

whereas the untreated authentic serine gave only one

spot which corresponded in position to the rapidIlymoving comlpcnent. The cause of this clhange in Rf

value is not clear. The recovered anmino acid, ifchromatograplhed with butanol: acetic acid: wvater(4: 1: 5 v 'v), gave only one spot, and this had theexpected Rf value for serine.

The compounds in the acidic fraction were sepa-

rated by gradient elution with formic acid on 200 to400 mesh Dowex-1-X-10 in the formate form (3, 18).Fractions of 3 ml wxere collected. The eluate in eachtube w-as evaporated to dryness and aerated to re-

move traces of formic acicl. The residue in eachtube was redissolved in 1 ml of water, a 0.1 ml aliquotwas removed for the determination of radioactivity,and the rest was titrated with standard NaOH withphenol red as indicator.

Degradationt of Glucose and Other Comipounds.The isolation of glucose by paper chromatographyas well as the acid hydrolysis of sucrose and the sub-sequent isolation of the glucose moiety were per-

formed according to the procedures described pre-

viously (24).Glucose w-as degraded with Leuiconostoc inesen-

teroides by the procedure of Gunsalus and Gibbs (8).The subsequent degradations of the fermentationproducts. ethanol and lactate, were performed as

described by Canvin and Beevers (5).The carboxvl carbon of glycine or serine was

determiniedl by ninhydrin decarboxylation (7). Thetotal carbon of the water soluble compounids was re-

coveredl by persulfate oxidation ( 10).Total carbon was determined as BaCO... bhit its

radioactivity -\as measured with the liquid scintilla-tion mletblod. BaC1403 wvas treated with acid and theC'40.. formiecl was trapped in 8 ml of solution (ethan-olanmine 0.2 ml, & methyl Cellos'olve 7.8 ml).

Assay of Radioactivity. The radioactivity of all

saml)les was determined with an automatic Packarlscintillation spectrometer with wvindow settings of10 to 50 anid 50 to 100. The liquid scintillator system

for water soluble compoundIs consisted of 60 g naph-thalene, 4 g 2,5-diphenyloxazole, 0.2 g 1.4-bis-1-(5-phenyloxazolyl)-benzene, 100 ml methyl hydrate, 20ml ethylene glycol, and p-dioxane to make a finalvolume of 1 liter (2). Amino acids and organicacids were analyzed by adding 0.1 ml fractions fromthe columns to 10 ml of this scintillator mixture.The scintillator system for C140 consisted of 2.75 g2,5-diphenyloxazole, 0.25 g 1 ,4-bis-1-(5-phenyloxazol-yl) -benzene, and 500 ml toluene. Ten ml of thisliquid scintillator were added to each 8 nml sampleof ethanolamine-methyl Cellosolve mixture contain-ing C'4O2.

Experimental Results

Metabolism17 of Glyciine-2-C14 in Kliapli If 7icatLeazves. Accordling to the postulated glyoxylate-serine pathway. glycine is first convertedl to serineby reacting with a one-carbon unit before its carbonatoms are incorporated into hexoses. The timecourse experiment with glycine-2-C14 (fig 1) showsclearly that this anmino acid w%as converted rapidlyto serine, which acquired 39 % of the radioactivityin the total amino acid fraction after 10 minutes,whereas glycine retained only 25 %. After 25minutes. serine possessed 29 c/c and glycine 12 %and after 45 minutes serine 34 %'- andI glycine 10 %.Alanine and aspartic acid gained radioactivity rela-tively slowly, and glutamic acid became radioactiveeven more slowly than aspartic acidl. A group ofninhlydcrin positive compoun(ds eluted early from theion-exchange resin columnli became labeled quicklyand possessed about the samiie radioactivity as glycineafter 10 minutes. In this group of compounds, onecomponent has been tentatively idlentified as phos-phoserine. (Identification will be described morefully in a later paper; the compound contained phos-phate and was ninhydrin positive. It was eluted fromDowex 50 at the sanme position as phosphoserine. hadthe samiie Rf as phosphoserine on paper chronmato-grams developed with ether: formic acid: water, andyieldecl serine upon acid hYdrolysis.) The amountof raclioactivity in these unknowns, including phos-phoserine. decreasedl with time. The reduction ofradioactivity was evident in all anmino acids after 45minutes. These results clearly show that glycine israpidily converted to serine.

Metabolismii of Serine-l-C14 ini K/iapiph 'heatLeaves. The interconversion of glycine and serine,which has been established in animlal tissues anidmicroorganismis. often has been assumiied to occuralso in plants. If interconversion occurs, serine-1-C'4shoull X ield C14-labeled glvcine. Figure 2 showrs re-sults fronm a timle course experiment wvith serine-1-C' 4. Contrary to some opinions, the formation ofgly-cine from serine does not take place readily invivo. Glycine had 1.3 %, 0.5) %, and 0.7 % of thetotal radioactivity in the amiiino acids after 10, 25,and 45 minutes, respectively. These results suggestthat the conversion of serine to glycine is very slug-

431



PLANT PHYSIOLOGY

0

.4

K

i

z

4

0

FRACTION NUMBER

50FRACTION NUMBER

50FRACTION NUMBER

FIG. 1. Distribution of C14 inamino acids isolated from wheatleaves fed with glycine-2-C14 for:(A) 10 minutes, (B) 25 minutes, and(C) 45 minutes.

432

IV 0

K

:i

C)

4

1c

P.,

C)

o L

Glyctne-2-C14Time: 45 min.

C

-a

go~~~~~~~~L0~~~~~~0

_ uz~~

c

Iv

Z a1~~~~~~~.10010



WANG & BURRIS-METABOLISM OF C]4-SERINE & GLYCINE IN WHEAT

uz0

e

3C.;

C.)

10 50FRACTION NUMBER

FIG. 2. Distribution of C14 inamino acids isolated from wheatleaves fed with serine-1-C14 for: (A)10 minutes, (B) 25 minutes, and (C)45 minutes.

SERINE-1-C14Time: 25 min.

5 -

04 . t

4 0B

3 1

2

1J ~ ~ *1~a

433

100

UZa

C.;6

.4'43

C)i1*4

m 2

41

UN44K

>4

9-:



PLANT PHYSIOLOGY

FRACTION NUMBER

FIG. 3. Distribution of C14 inamino acids isolated from wheatleaves fed with serine-3-C14 for: (A)10 minutes, (B) 25 minutes, and (C)45 minutes.

434

10

0

x

C.a:

0;

6

z

C-

4:5:0a

10

x

z

E-

< 5Oa

FRACTION NUMBER



WN'ANG & BURRIS-11ETABOLISAI OF C14-SERINE & GLYCINE IN W'HEAT

gish, and that even the small amount of C14 in glycinemay not all have come directly from serine. Thiswill become evident later. Alanine and aspartic acidbecame labeled only slowly, and glutamic acid gainedradioactivity much more slowly than aspartic acid.The group of unknown ninhydrin positive compoundsincluding phosphoserine acquired radioactivity veryrapidly and had approximately the same amount ofisotope as serine after 10 minutes. Their relativeradioactivity gradually decreased with time.

After fractions from the amino acid analyzerconstituting the serine-threonine peak were pooled,these two compounds were separated by chromatog-raphy on paper. It was judged from the radioauto-graphs of the chromatograms that threonine con-tained about 10 to 20 % of the total radioactivity ofthese two compounds.

Metabolismji of Serine-3-C14 in Khapli WheatLeaves. The metabolism of Khapli wheat, describedearlier by Wang and Waygood (24), was very simi-lar to that of the varieties listed in table III. Fromthe interconversion reaction of glycine and serine,the ,8-carbon atom of serine should yield a one-carbon unit and the rest of the molecule glycine.Glycine arising directly from serine-3-C14 shouldnot contain any C14. However, in a time course ex-periment, the radioactivity found in glycine was0.5 %, 0.8 %, and 0.8 % of the total radioactivity ofthe amino acid fraction (0.3 %, 0.5 %, & 0.4 % ofthe total radioactivity) after 10, 25, and 45 minutes,respectively (fig 3). The amounts of C14 in glycinederived from serine-3-C14 or from serine-1-C14 wereessentially the same after each time of exposure.

Metabolismji of Glycolic Acid-2-C14 in KliapliWheat Leaves. Glycolic acid-2-C14 was incorporatedreadily into various amino acids and gave a labelingpattern similar to that from glycine-2-C14 (fig 4).Serine again contained the highest amount of the iso-tope. The results differed, however, from glycine-2-C14 feeding because much more C14 from glycolicacid-2-C'4 was incorporated into glutamic acid thaninto aspartic acid.

Distribution of C14 Amiong the Organic Acids.Glycine and serine were rapidly metabolized by wheatleaves. A considerable portion of the C14 from thesecompounds was transferred to organic acids in 10minutes. The total alcohol soluble less chloroformsoluble material carried the following percentagesof their radioactivity in the organic acid fraction:17, 16, or 12 % after 10, 25, or 45 minutes, respec-tively, when serine-1-C14 was the substrate. It was16, 29, or 15 % after the same periods when serine-3-C14 was the substrate.

Analysis of the organic acid fraction revealedthat glyceric acid always contained the highestamount of radioactivity after 10 minutes. As thefeeding time increased, malic acid became the mostradioactive organic acid. Another compound, whichhas been tentatively identified as a guanine deriva-tive, accumulated only slightly less C14 than did malicacid. Surprisingly, citric, fumaric, and succinic acids

0

x

6z.

:uH

0_4:

10 50 100FRACTION NUMBER

FIG. 4. Distribution of C14 in amino acids isolatedfrom wheat leaves fed with glycolate-2-CI4 for 40 min-utes.

acquired very little C14 even considering ihleir poolsizes. The pool size of the tricarboxylic acid cycleacids was rather constant and probably was less thana tenth the concentration of the amino acids.

The glyceric acid peak contained glvcolic acidand an unknown acid. After resolving thesc threeacids by paper chromatography, it was found thatglycolate and the unknown acid possessed approxi-mately 10 to 20 % of the total radioactivity of thesethree acids.

Intramolecular Distribuition of C14 inl VariousMetabolites front Serine-1-C14. Table I shows thepercentage distribution of C14 in glucose whenserine-1_C14 was used as the substrate. Carbons 3and 4 contained nearly all of the C14 in free glucoseafter 10 minutes; the glucose from sucrose possessedmuch less C14. Nevertheless, carbons 3 and 4 stillcontained the highest concentration of isotope,57.4 %. The concentration of C14 in carbons 1 and6 increased with time. The distribution of C14 pre-dominantly in carbon atoms 3 and 4 of glucoseclearly suggests that this sugar was formed by acondensation of two three-carbon units.

Examination of the internal distributions of C14revealed that C14 was predominantly in the carboxylcarbon of serine but not of glycine (table II). Thecarboxyl carbon of serine had 83 % of the total radio-activity, whereas that of glycine had only 13 %.

435



PLANT PHYSIOLOGY

Table I

Percentage Inutramlolccul(lr D)istributiou of (C'4 Based o)tSpecific Radioactizvity ir flic Gliucose Mlloicty of

Sutcrose Derived fr oini Serine-l -C' 4 Feediig

Feedingtime(min) Ci

10*102D

45

2.41(1.510.315.2

" Free glucose rec

rather tllani glufree glucose hla(dBaCC 3 and the200 ,umoles.

Table II

Perc)ultage iititra;; olecu-lar I)istribu tiou of ('14 1J-ase(l on

Specific Radioactivitv in Glyciue , SCruic J)crizvd

from Scrine- -C14 FcedingThe labele(d serinie recovered xwas (lilute(l wxith 32.2 imig

Positioin of carboui of glucose of unlabeled serine. C-1 ( decarboxvlation by uinhydriuin the serime isolate(d liad sp)ecific activities of 1,040, 320),

2;> t ¢; aud 200 cpm mg BaCO., at 10, 25, ancl 45 miuiuites, re-

8.9) 39.5 44.1 4.5 0.6 spectively, and the BaCOQ froimi persulfate oxidati(u)l8.2 31.9 25.5 11.5 4.1 (0C-1,2,3) ha(l 410, 150, and( 120 cl)m mg. resj)ectively.

Treatmlenits xx-ith ninhIildriin or persulfate wx ere rull in

15.2 20.2 31.5 9.1 5.6 duplicate xxith each 10, 25, and 45 iminute sampl)e carry-__ _ inlg 11,200, 4,300, anid 3,560 cpm, respectively. Ihle label-overedl froumi the leaxves wxas (legraded edgive recovered xas (lilute(l xith 32.2 uf un

Icose derived fromIl sucrose. C-4 in labeled glycine. The C-1 in glyciine gave BaCO., froma specific activity of 2,6)50 cpwm mlig ninhydrin treatmenit ith 17, 3, andci 12 cpmi mig BaC()

amounit of glucose xas approximately at 10, 25, and(I 45 minlutes, respectivelv, sndl the BaC()(lerive(I fronm persulfate oxi(lationi hla(d 70, 6, and 15 cpmlmg, resipectiv ely. Treatmlenits itlh ninhydrin or Ipersul-fate were runl in dupllicate with eachl 10, 25, anid 45 mmn-ute samiple carrying 2,020, 220, and( 450 cp)m, respectively.

C in the a- and 8-carhns (of serine lncreasel from

17 (c( after 10 miniiutes to 43 after 45 miniultes. A

similar increase of in tlle carbhoxl carbon ofglvcine also cas evident, anid the radioactivity ratioof a-carb)on to cearoxy l carbon of g,2I1Ycille gra(ltllall

aIl)roacheed unity.lIet(blisbl i of (Nvciuei --C(' i,n Leaves of (uriouMs

1T'lieat 'arietics. Tlhree wheat varieties, Lee, Rtis-sell, and Henry, xere studie(d to ascertain wh-llether

varieties other tllani Khlapli also efficienitlx utilizethe carhon chain of -lvNcine for the sy-nthesis of

Feedingtime(min)

102545

(lycile

COOHa-carbon* CarHcarboni

877559

1325

41

Serinie

aandl/ ( ()OHcarbons carbon

172643

837457

Radioactivity- in C-2,3 of seriie anid C-2 of glxciniexvas calculated h! nlifferenice between the C"' recover-ed by persulfate anId by ninhvdrin treatmenit.

0

K

5

0.

1-9

0

x

67

>,I-'4

E-ua-'0

Q

u20 40 60 80 u 20 40 60 80

FRACTION NUMBER FRACTION NUMBER

Fi(,. 5. D)istribution of C'4 in organic acids isolated from xxheat leaxes fe(d xxith A ) serine-14 '', ( 1' ) serile(-3 C'14 f)1- 10 mi nutes.

436



WN'ANG & BURRIS-METABOLISM OF C14-SERINE & GLYCINE IN W'HEAT

Table IIIMetabolis1,n of Glycibe-2-C14 in Leaves of Lce, Ruissell, & Henry IT'heat

Feeding period was 30 minutes.

Wheat VarietyFractioni

Lee Russell Henry

cpm* % cpm* % cpm* %Alcohol soluble

(less CHCl3 soluble) 1,099,900 IAmino acids 514,000Organic acids 253,000Sugars 229,700* cpm per 30 leaves having a dry weight of 0.24 g.

100472321

1,187,000479,500221,300349,000

100411929

1,245,000552,400173,100352,500

100451428

sugars. GlIvcine-2-C14 was metabolized rapidly byall three varieties (table III). After 30 minutes,21 %V, 29 . or 28 % of the total radioactivity in thealcohol soluble less chloroform soluble material ap-peared in sugars in Lee, Russell, and Henry varieties,respectively. Essentially all radioactivity in thesugar fraction resided in glucose, fructose, and su-crose. Only the amino acids recovered from the ex-tract from Russell wheat leaves were analyzed.They had a distribution of C14 similar to that fromKhapli wheat. Among the amino acids, serine againhad the highest concentration of C14. Serine andalanine had 35 % and 11 % of the total C'4 in theamino acid fraction, respectively, whereas glycineretained only 9 %. Paper chromatography of theorganic acids from Russell wheat leaves revealedthat glyceric acid had much more C14 than did gly-colic acid.

DiscussionIn prior investigations of the transformation of

glycine to sugars in wheat leaves, a pathway ofglyoxylate-serine metabolism was proposed (23, 24),and a similar pathway also has been described byRabson et al. (19). Although evidence reported sofar has favored the operation of this pathway inwheat leaves, a number of questions previously couldnot be resol-ed wNithout further experimental evi-dence.

In mlammilalian tissues (16, 17) and miiicroorgan-isnms (4), gl-cine is oxidized to CO, and formatethrough glyoxylate. This often has been assumed tooccur in plants, because both transaminase (15 &WN'aygood. personal communication) and glycolic acidoxidase (6, 21) are present in plants. However, inisotopic competition experinments writh -wheat leaves(24), neither glycolate nor glyoxylate lowered theC'4 in sugars when glycine-C14 was the substrate.On the contrary, both compounds enhanced the rateof transfer of C14 from glycine-2-C14 to sugars, andthe formation of sugars from glyoxylate-C'4 wvasgreatly reduced by the addition of glycine. Further-more, no difference was found between the radio-activity in glyoxylate derived from feeding with

glycine-2-C14 alone and froml glycine-2-C14 plujsglyoxylate. The present evidence shows clearly thatglycine-2-C'4 was converte(d rapidly to serine (fig 1)in vivo in wheat leaves and gave rise to highly radio-active glyceric acid but only slightly radioactiveglycolic acid. Glycolic acid-2-C14 also was quicklyincorporated into glycine and serine (fig 4). It wasapparent that either glycolate or glycine was con-verted rapidly to serine, and that the oxidation ofglycine via glyoxylate did not constitute a majorroute.

The formation of serine from glycine requires aone-carbon unit. McConnell and Bilinski (13) and(Tolbert (22) demonstrated that formate could serveas a source of the /8-carbon of serine in wheat andbarley plants. The slow conversion of glycine toglyoxylate (24) makes it difficult to visualize howglyoxylate can serve as a source of one-carbon unitsfor the rapid synthesis of serine. The extent of con-version of the a-carbon of glycine to the ,8-carbonof serine is reflected in the amount of C14 in carbons1 and 6 of glucose formed from glycine-2-C'4 (24).The mechanism of direct cleavage of glycine, whichgives rise to a one-carbon unit and CO, (20), isquite attractive as an alternative route. Althouglhno direct evidence supports this route, it warranitsinvestigation.

The metabolic conversion of serine to glycineand a one-carbon unit is reversible and has beendemonstrated to occur in animals (12) and plants(26). MIcConnell and Findlayson (14) have indi-cated that wheat plants also effect the conversion.If so. serine-1-C'4 should transfer its label readilv toglycine. However, the very low radioactivity foundin glycine (fig 2) indicates that the conversion ofserine to glycine is very sluggish in vivo in wheatleaves; even the small amount of C'4 found mlay notall have come directly from serine. This conclusionis supported by the fact that glycine produced afterfeeding serine-3-C14 (fig 3) possessed essentiallythe same amount of isotope as that from serine-1-C'4,although according to the postulated transfer meclh-anism carbon should be transferred from the 1 butnot the 3 position. Furthermore, the isotope in gly-cine derived froimi serine-1-C'4 was predomlinently

437



PLANT PHYSIOLOGY

locate(l in the a-carbon r1atlher than in the l)re(lictedcarboxyl positionI (table IF). Serine fromil .he sanieexpelimilenlt, however, was still predominantily labeledas exxpecte(l in the carb)oxyl group) (table II). Ev1i-dentlv, the C(4 in glycine resulte(d fromil the re-entryof C(4 fromli serine into the gl-oxylate-serine path-wvay rather tlian from (lirect conversion of serine toglvcine. Tlhele appears to l)e a (liscreplancy betweenthe findings of MIcConnell aan(l F1ndlavson (14) andthose reporte(d here. They observed a high concen-tration of C' in the caarboxvl carblon of glvcine re-covere(l froml protein hlen n.,-serine-l_C'4 -vas sup-plie(I to wheat in a long termii exIperiment; extensiverecycling of the isotope fromii the substrate throughglvr-oxylate-serine patllway could( have occurre(l (lur-ing the long exposure l)eriod.

The intramolecular distribution of C'4 in glucosepro(luce(l by leaves fed w-ithlvarious C' -labeled tw-o-carbon comlpounlds suggested thiait glucose xws-ias rm-e(l frolim a con(lensation of two three-carbon units(24 ). In this transformation. serilne was thougIltto be the first three-carbon precursor of glucose.More conclusive evidence to suIpport this hypothesiscamile -fromol the pireseilt studly of the intramolecular(liitril)utionl ef C'4 inl ,lucose fromii serine-I1C' .The isotope was as expectedl pre(lomilnantly locatedin carbon atomiis 3 and 4 of glucose (table T). Re-centlv . Jiminez et al. ( 9 ) rep)rtedl a plredominiantlabelilng of carbons 1 and 6 in iglucose fromil sel-ilne-3-C'.

The percentage of total activity in carbons 3 and(4 in free glutcose recovered 10 mlinutes after fee(ingserine-1-C4 Xwas 83.6 ', andl in the glucose mioietyof sucr-ose recovered after 10, 25.and 45 miniuttes xwas57.4 ',. 60.7 , anld 51.7 (( resl)ectivelv. The car-hoxvl carbon of serine carried 83 c. 74 'j. an(l 57 %of thle C14 in the nm lecule after the s.ame intervals.These values are in reLsonablhV goodl agreement w-iththose of glucose. It seemils evident that the carbonchaini of serine is incorl)orate(l into glucose essen-tiall!V 1un1altered.

The ran(lomlizationi in carbons 1, 2. 5 ai(l 6 inglucose is likely the resuilt of the photosynthetic re-entr-y of C'4.0 (lerived froml the oxidation of serilne.The imlost conclusive evidence shox-ing, the re-entryof C'. coniles froml experimienits xvithi serine-3-C'4,because all C' incorporated iiito glvcine has to comiefr'on- a route or routes other than the (lirect conver-Sionl of serine to glycine. The predlonminant dlistribu-tioll of C'4 in the a-carbon of glv\cine fronm serilne-1-C'4 also n'av he consi(lere(l as unequivocal evideencefor the recvcliigr of the isotope. The extent of rail-domizati,mn in carbons 1. 2. 5, an(l 6 in glucose is.tler-efore, (lelenent up)oln the (legree of labeling inthe a- and fl-carbons of seriine xvhich in turin is (le-penl(leilt upoIn thle (legr-ee of labeling in the a-carbonof glv cine.

\V anlg alnld \Waygood (24 repor-te(l ai1 une(qualleg-ree of randonmization betwx een carlons 2 and 5S

or carbolns 3 an(l 4 in glucose dependling Ulupon w-hetherthe substrate glycinie wxas labele(d in the 1 or 2 posi-tioIn. The present evidence suggests that this inl-e(luality of C14 (listribution arises becal-use tlhe j'14fromii serine-1_-C4 goes preferentially inito tle a-carboln of glvcinie (table II). These observationlsiiiake the hypothesis of a (lirect cleavage of glv-cinieto a onie-carbon uinilt and(I CO., highlyl pIlausib)le. be-cause in this process tlhe a-carbon of g-lycine is asource of the fl-carboni of serinie and tlhe carboxv lcarbon yields CO., vhich may reelnter tlhe glyoxylate-seriI4e p)athwvay pre(lonlinantly in the a-carboni ofglycinie. However, the miechaniism that pro(duicesglyci'ne vhich i's prefereiltially labeled in the a-posi-tion is not at all clear.

The rapid labeling of llhosphoserine x llen glvcol-te-2-C'4, glycilne-2-C, serine 1 C'4. or serine-3-C''vas fe(d to xvheat leaves is of initerest. D)ecisioll re-,gardHig, the plarticipatioln of tl,is collpoullnd in thleglvoxvlate-serinie pathlway as ain iilterIue(diate axw aitsfurther- investigatioll. Nevertheless, the fact thlatlabeled substrates vere coIlverte(l rapidly to lilos-phoserine suggests tlhe likelihoo(I of its palrticipatiol.

Summary\Vlheln glvc,late-2-C '- was -fedl to xw heat leaves.

it xwas coivxerte(l rapidly to gglvciie and(I ser-ilne. Ser-imie wvas formiie(d rapidlyN fromii !gl1vcine-2-C-' and1()s-sesse(l 39 ' of the total radioactivity ini the aimlinoaici(l fraction after 10 minutes, xhereas glvcilleretaine(l only- 25 ((; after 25 minuittes, se-ille Iad29 % in( glvciine 12 q. In conltrast, little gl cin1ew as fol-rmie(d fr-omll seriie-1-C'4'. and1( it possessed o1l1y1.3 (( 0.(5 (,. or 0.7 o(f the total radioactivitv ot(the aiiiii-o aci(lS after 10, 25, or1 45) milillltes, respec-tivelv. Serinie3-C' vI xvas niot expecte(l toield laleled glx-ciie by- direct coiversion. (ax e

gl-ycine xN ith the samiie amoiiunt of C4 as that rl-Oserine-1-C14. Furtherimiore, thle itramolecular (lis-tribution of Cl-' in glycine pro(luced from serile-l-C14 sowved a major portiolI of the isotople residli11in the a-carboni of gl-cille rather than in the exp)ecte(lposition in the carboxyl carbon-. Apparenftlv glyci ieis syIlthesized by one Metabolic pathway ald oxi(lize(dby- anotlher.

Anialysis of the organic acid fractioni foriie(d lbvx-hecat leaves froint glycine-2-C' ', seriine-l-C'I-, aindserine-3-C" shoxved that glvNceric acid xvas labeledquickly and p)ossessed the lighest radi-oactivity after10 milinutes of exposure. Much C'' xvas .transferredto pliosploserilne froill serine-1C'4, seriine-3-C' ,

gl-cine-2-C14, and glycolate-2-C' 4.These results, as xx ell as the ilitraimlolecular (lis-

tribution of C'" in glucose pro(lucedI frolmi serine-1-C14, agree xxitl the previously plroposed pathxvay ofgh voxvlate-serine letabolisn all(n clearl- (lenmonstratethe iillportalnt r-ole of seriiie in glyciie imetabolisill inxw lheat leaves.

438S



WANG & BURRIS-METABOLISM OF

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carbon metabolism of c14-labeled aminoacids …carbon metabolism of c14-labeled aminoacidsin...

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