Propionate in Heme Biosynthesis in Soybean - Plant Physiology

Plant Physiol. (1966) 41, 1673-1680

Propionate in Heme Biosynthesis in Soybean Nodules'Earl K. Jackson2 and Harold J. Evans

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331

Received September 6, 1966.

Summary. When soybean nodules are incubated with propionate-2-14C the hememoiety of leghemoglobin becomes labeled. The incorporation of propionate-2-14Cinto heme is linear with time and it appears that propionate is utilized without a lagperiod. The rate of incorporation of propionate-2-14C into heme is more rapid thanthe rate of incorporation of succinate-2-14C and citrate-1,5-14C, however, these ratesof incorporation may be influenced by different sizes of endogenous pools of organicacids.

Additional radioactive tracer experiments demonstrate that the supply of hemeprecursors from propionate is competitive with the supply of heme precursors fromthe citric acid cycle. When the concentration of propionate was high in the incuba-tion mixture, the rate of succinate-2-14C incorporation into heme was decreased.Furthermore, when a large amount of succinate or acetate is added to the incubationmixture containing whole nodules, the rate of incorporation of propionate-2-14C intoheme is reduced. The data support the hypothesis that propionate utilization makespossible a mechanism for the formation of succinyl-CoA in addition to that providedby the citric acid cycle.

The fact that propionate is readilv utilized by bacteroids suggested that thiscompound may be a normal metabolite in nodules. No detectable pool of propionatewas found, however, in either soybean nodules or in isolated bacteroids suggestingthat propionate, if present, is utilized as rapidly as it is formed. Experiments inwhich cell-free extracts of nodule bacteroids were used demonstrated the conversionof lactate to propionate. The cofactor requirements for these enzymic reactions areadenosine 5-triphosphate, Mg"+ and reduced nicotinamide adenine dinucleotide.

Cobalt is essential for Rhizobiunm species andsymbiotically grown legumes (2,16, 17, 18) but hasnot been shown to be essential for either leguminousor non-leguminous plants per se. The specific roleor roles of cobalt in the metabolism of either sym-biotic or free-living nitrogen-fixing organisms hasnot been adequately determined. De Hertogh et al.(10) have shown that propionate is activated in thepresence of ATP and CoA and that the produict,propionyl-CoA, is carboxylated yielding methyl-malonyl-CoA and finally converted to sticcinyl-CoAvia the methylmalonyl-CoA mutase reaction. Cobaltdeficiency in Rhi_kobhiotn cells is known to resuilt ina decreased synthesis of B,2 coenzyme which im-pairs the activity of the methylmalonyl-CoA mutasereaction (10). This lesion, apparently is respon-

1 Technical paper No. 2189 of the Oregon Agricul-tuiral Experiment Station. This research was supportedin part by grants from the National Aeronautics andSpace Administration [NsG(T)-68] and the NationalScience Foundation (GB2518).

2 Present address: Central Research Departmenit. E. I.dti Pont de Nemours and Company, Experimental Sta-tion, Wilmington, Delaware.

sible for the failure of cobalt deficient Rhir-obiuincells to effectively metabolize propionate.

There is evidence suggesting that vitamin B12is involved in the biosynthesis of hemoglobin inanimals (4, 7, 11), however, knowledge of the pre-cise sites where the vitamin functions is incomplete.An involvement in some way of vitamin B12 in thebiosynthesis of leghemoglobin was suggested by thedemonstration (2) of a significant reduction inthe content of nodule leghemoglobin when soybeanswere cultured in media lacking adequate cobalt.Fuirther, it has been shown that (15) the rates ofincrease in concentrations of both B12 coenzymeand leghemoglobin are nearly parallel during thedevelopment of sovbean nodules. Wilson andReisenauer (25) have shown that the nodules ofsymbiotically-grown alfalfa contain only traces ofleghemoglobin when 0.001 ,ug of cobalt per liter wassupplied in the nutrient meditum. When 0.010 ugof cobalt per liter was provided to alfalfa plants,the leghemoglobin content of the nodules increasedstrikingly.

By the utse of specifically labeled glycine-'4Cand acetate_14C, Richmond and Salomon (21) havedemonstrated that the general sequence of reactions

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leading to heme formation in soybean nodulles is

similar to that established for hemne sv nthes;sanimals. The pathway of acetate ntilization ill

porphyrin s nthesis is thronigh the citric aci(l cycle

wvhich leads to suiccinyl-CoA formation. Snccil-I 1-

CoA is theni coii(leinse(l with glycile to form-l (lelta-

alminolevotlinic aIcid, the (direct precu rsor of

p)orphyrtil s.

Ini soy1bean nodnles the acti vati(all of propliollate"nll coiwersioni to sluccinyl-Co. \½ ila methylnmilalolyl-

CoA½ makes possible a second( miiechlisaim fol- the

formiiationi of stccinyl-CoA ((), 1)). The p1)1-p)oscof this investigationi is to (lete-miilie Oietlier pro-

pionate niay serve as a signlificaiit precnrsor tofohenie biosynthesis as well as for participation in

citric aci(l cycle. Since nlothinlg is knox\x abont

propionate biosynthesis in leginimie niodoiles's, it \\xas(lesired to obtain in formation onl possille pathiw\\lys

of propionate formatioll.

Material and Methods

Soi rcc of Chemticails. Recrystallized hemin,

GSH anid the (lipotassiuii salt of \TP wx ere o)-

taimed from N\itritional Biochemicals Corporationl.

Sodium propionate-2-14C (5.15, mc/nimole), citrate-

1,5'4C (5.32 mc/mmole a-ketoglnltaate

(6.94 nic/mmole), sniccinate-2-24C (').1 mic/mniole)

ai-(d a-aminoleviflinate-4- IC (11.7 mnicmmole)

were pturchase(d froni New\ England Nuiclear Cor-

poration and sodinim lactate-l-'C ( 5.47 mc/mniole

an(I lactate-2-14,C (11.7 nic/niole ) were obtained

from the V"olk Radiochemical Company. CoenzymeA was puirchased froni the Signia Chemical Con-

paniy aid(I other cihemicals ain(d solvents ulsed were

reageint grade from commercial souirces.

Biological Matc('ri(ls. The biological materials

utilized in these experiments were soybealis (Glvcincazax Merr. variety Chippewxa) an(l coxxwpeas(rI Wna

sinensis L. variety Ironi clay misxe( ). The plants

were grown in a greenihouise1lsilig the ciltiural

methods dlescribedl by Ahmed and( E\ans (1,2).

Saltsuised in theniutrient soluitioni werenot puirified.Theno(itiles were harvested when plants were 38

to 48 days old. Precise ages ofno(dIules areindi-

cated in the legenidis of figuires andI tables.

rati.on,( anid Ieac1if1icalit of Orqa ic A4 cids.

The ether extraction method of Swin andtUtter(23) was

s

itilized for- the colutmin chromatographicseparatioian ideintificatio ofnon\volatile organiic

acids in odIuiles anid extracts. The idenitities of

stccinate, fumarate andmmalate that were isolatedl

from a Celite colimn were conifirnied by co-chro-

matography of these acids with known standards

on thin-layer plates of silica gel G (24). For the

separation of acids in nodIule extracts by thin-layer

chromatography, the method of Svim anid Utter

(23) was modifiedI by passing the water extract

of nodules, after evaporation of the ether, throulgha Dowex 50WA X8 cation exchange columnm to re-

moVe almino aci(ls. The eluiate froni the coltimas evap)orate(l to dryness and taken ip ml

of 9 % ethanol. A 100M)1 alicllqot wvas applie(d toglass plates (24).

lor the (leterulillatioll (f the volatile fattYacids, f-eslh n11'dule were Illacerated ini a mortar

with ani equal v-olmie of ater. Flacteroi(ls fromilnoduiles were Tiiixe(I with 1 nil of xater and brokenafter fr-eezing by uise of (ain Eaton lpress. The

Iti(Iniogel ates of (i1ul1es (or 1) cteroi(ls were aci(lifie(dto p1 2 with 1. S( ) aad s1uil jected to steaim (lis-tillattion. rhe (listillate was nieuitralize(d to pl4wx ith 0.2 Nx Na( ) 1t1andten evaporatedl to niear

d1ryless i a rotary evapl)rator. The resi(dlue fromCeaCh1 Saniil)le' xxLS diSSOlVed ill 1.5 nil of 0.2 N I1., 0,

anl applied to a Cel ite col mini (23 for isolatiol (Ifthe acids. ut\xric and propionic acids were eluite(dxvith 1()() % chlor-oforni-, acetate xxith 95 % chloro-fornm and S% l-out\xl alcolhol, an(l formate vitli90 % chloroformii and 10 % -b1lutyl alcohol. Theidentity of pl-pl)ioll1ate xxVas fLrther- established 1bu1se of propion<ate-2-'4C. One tc of this compolndl(Iwxas ad(lledl to a standard mixtuire of volatile acidsanI(I it was showvn that the peak of radioactivitycorrespon(le(l xvitli the propionate peak as deter-minied by titration xxith NaQI I.

Isolationt and Parific tiom of IIcuiic. TIhe nietiodlise(l for the isolatioln anL(l plni ficatio0i of henie

froni leguinie n1o(dulies was developed fromn thieniethoIs(lescribed b)y Richmonmd, Altmnia and Salo-nioni (20 ), Chii and(1 Cliii (6), Kiese and Kiur-z (14.an(l Tltlcher an(d Vishniac ( 13). Nodules wxere

washe(l thoroighly and then mnacerate(l in a niortarwxith an eiual voluime (If xvater. Theresi(lule xwas

renmove( by centrifuigationi aui(l the sLuperniatalnt frac-tioni xas adj nsted to p1i 3wxithi glacial acetic acid.

Siifficicent acetolle was ad(l(le to the supernatantsoluitioni tomake it 80 % acetolne. The precipitatevas removed by cenitrifulgatiom anl 20 mlof chloro-

form xvere addied to the aquleous acetone soliltioni.A.fter 2 houlrs atroonii temperature, the hemewasextracte(l thoroughly wxith 2(0ml aliquots of chloro-form. A fter combiined chlol-oformi extracts were

xvashed 4 times wviti 100 ml aliquots ofdistilledlvater, the chloroforni phase was evaporatedl to

dryness anid the residud1 e takeni uipill 0.5 ml pyridin.e.Chroniatographxofa 100) p.l sample of thismnaterialrevealed a fluorescenxt spot uder uiltra-violet lighltthat is typical of porphirins. Inordeer to remove

porphyripyns a other contaniiniiants froni the hieme

sample pyridine, suifficienit water to niake a %

pyridhlie sollti isa(l(le(l anmd the solutionchcroiiiatograhlieon0 a siliconie-impregnaatedIelllllosecoIl nin The col nin as ashed several

times xith xvateranc d the heme was eluited withpyridine-propanol-w(ater (1:3:12.5) v/v. Thesol-vent suibsequiently was removed by evaporation and

the heme xvas takeni uip in 1.0 ml pyridine andI use(l

for determination of radioactivity, for measuremenit

of heme content and for fuirther chromatography.The specific activity of heme isolated by thispro-

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ce(Il-c remaine(d essentially constant in 3 suic-

cessive chromatographic separations (table I).Fuirther proof that the heme samples were relativelypure was obtainedl by chromatography of a repre-

sentative sample of purified radioactive heme on

paper (6) and scanning the paper with a stripcouinter. In this experiment radioactivity was lim-

ite(l to 1 spot corresponding to that of a sample ofauithelntic heme. The spectrulm of the pyridlinehemocbromogeni from isolated samples was essen-

tially idenitical to that of recrystallized hemin.Further identification was accomplished by the(letermination of the RF valuies of heme samples inlutidinie and in water-propanol-pyridine (5.5 :0.1 :0.4)v/v (6). The concentration of heme was deter-mined by measuirement of the optical density oireduicecl pyridine hemochromogen at 555 my (12).

Alca(sutremiient of Radioactivity. For the meas-

uirement of radioactivity in the heme moiety ofleghemoglobin an aliqtuot containing 0.04 to 0.08,umole of pturified heme was placed in a glassplanchet and evaporated to dryness with a warm

jet of air. The samples were counted at infinitethinness uising a Ntuclear Chicago model 181 B gas

flow detector. The radioactivity was proportionalto the quantity of heme added to each planchet inthe range of 0.0 to 0.1 jumole of heme. The effi-ciency of the couinting procedure was 12 to 15 %and suifficient cotunts were recorded to obtain a

standard deviation of 1.4 % or less. In a typicalexperiment each 4 g sample of nodulles contained1.0 to 1.2 ,umoles of heme. The amouint of hemeisolated from each sample ranged from 0.42 to 0.58/Amoles and the incorporation of propionate-2-14Cand sticcinate-2-14C into heme was approximately1 % of that added to the reaction mixttures.

For the measturement of radioactivity in pro-

pionic and acetic acids the peak effltuent fractionfrom a Celite column was neutralized and an aliquotcontaining 0.2 to 0.6 micro eqtuivalent was added toa glass planchet, evaporated to dryness and countedat infinite thinness. In a single experiment 14CO.,was collected, converted into BaCO3, plated andassayecI for radioactivity at saturation thickness(19).

Standard Incubation Procedure. Nodules were

harvested, washed in ice water and samples of 4 g

fresh weight were weighed for use in isotope incor-

poration experiments. Each sample uinless other-wise indicated in legenids of tables and figutres wasplacedl in 6.0 ml of 0.1 M potassiutm phosphate butffer(pH 5.6). This pH was shown to be optimum forincorporation of propionate-2-14C in whole nodutles.To the 6.0 ml of buffer soluition were added 50/%moles gluitamate, 12 umoles glycine, 5 ,uc of 14C-labeled metabolite and 12 Mmoles of the appropriatenon-radioactive metabolite to minimize the effectof en(logeniouis pools of metal)olites. To aid in theintroduction of the reactants into the nodutle tisstues,each sample of noduiles immersed in the incubationmediulm was evacuiated at 70 mm Hg for 3 minutes.The ulse of boiled nodutle extracts in place of freshextracts in reaction mixtulres resulted in no meas-

uirable incorporation of radioactivity into the hemeof leghemoglobin and thus control reactions usingboiled extracts or noduiles are not presented foreach indlividlulal experiment.

Prepa(ration of Cell-Free Extr(1cts. Cell-freeextracts were prepared by freezing the noduiles illan equial voluime of 0.1 M potassiuim phosphate buif-fer (pH 7.0) in solid CO., and breaking the cellsin an Eaton press at a pressure of 8000 pouinds persquiare inch. 'fhe cell debris was removed by cen-

trifulgation and the extract dialyzed tinder argonfor 5 to 6 hours in 2 liters of 0.1 Ni potassitunmphosphate buffer (pH /7.0).

For the preparation of cell-free extracts ofbacteroids, noduiles were macerated in 2 voluimes of0.1 M phosphate buffer at pH 7.0, squieezed throuigh4 layers of cheesecloth to remove the solid debrisancI the bacteroids collectedl by centrifulgation. Thebacteroids were washed repeatedly with 40 ml vol-ilmes of 0.1 M phosphate btuffer at pH 7.0 until alltraces of hemoglobin were removed. A sltirry ofthe bacteroids (1 wt per 1 vol of 0.1 M phosphatebuffer at pH 7.0) was placed in an Eaton pressurecell, frozen in solid CO., and broken as describedfor nodules. After centriftugation, the cell-free ex-tract was dialyzed as described for nodtules andstored tunder argon at -70°. The protein deter-mination was madle by the Bil ret method (8).

Results

Orgainic Acids in Nodules. Since propionate isreadlily oxidized by the bacteroids from nodlulles and

Table I. Purity of Radioactive Heme Isolated from Coupea NodulesThirty-nine day-old cowpea nodules were infiltrated with 5 ,uc of b-aminol-evulinic-14C acid under the standatr(d

incubation conditions. After 12 hours heme vas isolated, purified and assayed by the procedure outlined in the Mate-rials and Methods section.

Times Heme Specific activitychromatographed' isolated of heme Standard

(No) (,moles) (cpm/4mole) deviation

1 0.54 27,900 + 2792 0.30 27,800 ± 2783 0.18 27,300 ± 273

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the enzymes for propionate utilization are presentin noduile bacteroids (10) propionate might beexpected to be present as a normal metabolite insoybean nodules. Investigations were carried out,therefore, to determine the content of propionateand(I other organic acids in nodtules. The data pre-sented in table II show that soybean noduiles containa relatively high concentration of acetic acid, meas-trable qtlantities of butyric and formic acids but nodetectal)le qtuantity of propionic acid. Even thoughradioactive propionate added to nodule homogenatescotldl be recovered satisfactorily, no evidence wasobtained by either coluimn chromatography or gaschromatography that propionate was present in soy-bean nodules.

Data in table II also indicate that nodules fromsoybean plants contain several citric acid cycle acids.Malic acid is present in the highest concentrationand appreciable amotints of a-ketoglutaric, stuccinic,fulmaric and isocitric acids were found. In thisanalysis no citric acid was detected, however, inother experiments a trace of this acid was fouind.The identity of the various citric acid cycle acidsisolated by column chromatography was confirmedby suibjecting a sample of each acid to thin-layerchromatography using the procedure of Ting andDugger (24). Thin-layer chromatography of nod-ile extracts consistently revealed 2 additionalacids with RE valtues of 0.09 and 0.02 btut thesecompotunds were not identified.

In another experiment, 4 g of nodules were in-ctibated with 5 ,uc propionate-2-'AC for 1 hour fol-lowing the standardl incubation procedlure. Citricaci(d cycle acids were separated by thin-layer chro-matography and plates were scanned for radio-activity. The restults showed that radioactivity wasincorporated into fuimarate, succinate, a-ketoglu-tarate and malate. These data confirm the observa-

Table II. Organic Acid Content of Soybean NodulesVolatile fatty acids in a 20 g sample of soybean

nodules (42 days old) and citric acid cycle acids in a 10g sample of soybean nodules (40 days old) were deter-minitied by the procedures described in Materials andMletlo(ls.

Acidl

Volatile acidsFormic

AceticPropionicButyric

Citric acid cvec a(cidsFu rtnaricSuccinic

a-KetoglutaricMalicCitricTsocitric

Concentrations(micro e(lulivalenitsper g fr wt)

0.6711.890.000.82

2.323.776.10

20.19(.O(2.90

tions ,of De Hertogh et al. (10) and( provide evidelncethat propionate may serve as an alternate carbonsouirce for maintenance of the citric acid cycle.

Incorporationi of 14C-labeled M1etabolites in1toHeme. From the (lata presented graphically in fig-tire 1 it is apparelnt that the heme moiety of leg-hemoglobin becomes radioactive when noduiles areincuibated with propionate-2-'4C. The rate of in-corporation of 1'C into heme from propionate-2-14Cis linear with time and appears to occuir withotut alag period. Similar results were obtained in otherexperiments using nodtules from cowpea plants.Sliced, homogenized or whole soybean noduiles, orcell-free extracts of soybean nodutiles, effectivelyincorporated labeledl propionate into heme but sub-jection of nodtules or extracts to 1000 for 3 minuitesprevented the incorporation.

Since propionate fuinctioned as a heme precuirsor,additional experiments were needed to determinethe rate of propionate incorporation into heme inrelation to the rates of incorporation of other metab-olites. In a series of replicate experiments therelative rates of incorporation of radioactive organicacids have been reproduicible and have shown thatpropionate is incorporated into heme at a fasterrate than either citrate or suiccinate ( fig 1). Iiiother experiments the rate of incorporation of 14Cfrom a-ketoglutarate-5-14C was slightly greater thanithat of propionate-2-14C and the initial rate ofincorporation of fuimarate-2,3-'4C was comparableto that of stuccinate-2-14C. In experiments com-parable to that described in figuire 1, -aminolevu-linate-4-1'AC was very rapidly incorporated inltoheme. The specific activity of the isolated hemeafter 1, 3, 5 and 7 hours of incubation was 1240,6700, 13,900 and 20,600 cpm respectively. This ratewas linear with time.

In an effort to avoid complications in interpre-tation of rates of incorporation of metabolites iintoheme resulting from different pool sizes of en(lo-genous metabolites, experiments were carried otittising cell-free extracts of soybean noduiles. Fromthe data in table III it appears that a-ketogltutarate-5..4C is convertedl into heme approximately 4 to 5times faster than either propionate-2-'4C or pro-pionate-1-11C. Decreasing the einzyme extract b)yone-half (table III Experiment ]) decreased therate of incorporation of propionate-2-14C into hemeby 40 %. The addition of 12 jumoles unlabele(dcarrier significantly redtuced the rate of incorpora-tion of propionate-2-14C or suiccinate-2,3-14C intoheme. Dialysis of the extract (table III Experiment2) resulted in a 2-fold increase in the rate ofincorporation of at-ketoglutarate-5-14C and lactate-1-' 4C into heme and increased the rate of incor-poration of b-aminolevtilinate-4-4C by approxi-mately 23 %. On the other hand, (lialysis failecl tosignificantly infltuence the rate of incorporationi ofpropionate-1-14C. The low rate of incorporationiof propionate might have been catisedl by lability

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g 1200-

G) 1000-.> a Propionate-2j4C>% E800-

tE 600- Citrate i1,Mc_U 400 /

°. 200-en Succinate-2 4C1 2 3~4 5 6 7'Time of incubation (hr)

FIG. 1 A comparison of the rates of incorporation ofpropionate-2-14C, citrate-1-5-14C and succinate-2-14C intothe heme moiety of leghemoglobin in intact soybean nod-ules. Each incubation mixture contained 5 ,Ac of radio-active metabolite and 12 jumoles of unlabeled metabolite.The Standard Incubation Procedure was followed andthe 4 g-samples of nodules were from plants 39 days old.

of enzymes involved in propionate utilization. Fromthe data in table III, it appears that lactate can alsofunction as a heme precursor.

Cootpeting Pathways for the Formation of Hemle

Precu rsors. Since a supply of sUccinyl CoA forheme synthesis could arise from either intermediatesof the citric acid cycle or from the utilization ofpropionate, it would be logical to expect that theaddition of non-radioactive propionate would resuiltin an increased supply of non-radioactive succinyl-CoA and thus dilute the incorporation into heme ofradioactivity from labeled intermediates of thecitric acid cycle. Table IV shows that the additionof 12 6moles of non-radioactive propionate to theincubation mixture resulted in a substantial redcuc-tion in the rate of incorporation of succinate-2-14Cinto heme. In 4 different experiments the trends inthe results were similar. Furthermore in reciprocalexperiments where 12 ,moles of non-radioactive suc-cinate were added to an incubation mixture therate of incorporation of I"C from propionate-2-l"Cwas reduced to an extent of 40 to 60 %.

The addition of non-radioactive acetate to eitherwhole or macerated nodules resulted in a decreasedlrate of incorporation of propionate-2-14C into heme.The effect of adding 12 /moles of non-radioactiveacetate to each reaction mixture on the rate ofincorporation into heme of propionate-2-l"C andsuccinate-2-"4-C is illustrated in figure 2. The addi-tion of unlabeled acetate reduced by approximately33 % the rate of incorporation of propionate-2-'4C

Table III. Incorporation of 14C-Labeled Precursors into Heme in Cell-free Extracts from Soybean NodulesExperiment 1. Cell-free extract was prepared from 40 day-old soybean nodules and dialyzed for 6 hours in 0.1 M

phosphate buffer (pH 7.0). The reaction mixture in a final volume of 6 ml 0.1 M phosphate buffer at pH5.6 con.tained 2 ,uc 14C-labeled metabolite; 10 ,umoles of ATP and MgCl9; and 0.2 ,mole of CoA and NADH.Twelve jumoles carrier were added as indicated. The incubation mixture contained 20 mg protein/ml unless indi-cated. Tubes were incubated 90 min at 350. Radioactivity incorporated into hemne using a boiled extract (2 cpm/0.07 pwmole heme) was subtracted from each determination.

Experiment 2. Same as in experiment 1 except that the cell-free extract was prepared from 28 day-old nodules.Each reaction mixture contained 0.26 pmole heme from extract and was incutbated for 50 min at 380 at pH 7.0.

Unlabeledcarrier Sp activit-added of heme

Metabolite Extract (jLmoles) (cpm/,Omole)Experiment 1

Propionate-2-'4CPropionate-2-'4CPropionate-2-14C!Succinate-2,3- 4CSuccinate-2,3-' 4Ca-ketoglutaric-5-1 4CLactate-2-1 4C

Experiment 2Propionate-1-14CPropionate-1-14Ca-ketogluttaric-5-3 4Ca-ketoglutaric-5-14Cb-aminolevulinate-4-' 4Cb-aminolevulinate-4-1 4CLactate-i-14CLactate-i-14CLactate-1-14C + 20 Atmoles

unlabeled proprionate1 Reaction mixture contained 10 mg protein/ml.

dialyzeddialvzeddialyzeddialyzeddialvzeddial! zed(lialvzed

cruide(dial Xzedcrutdedial]-zedcrutdedialyzedcrudelialyzed

dial,zed

012001200

1212121212121212

4582112701316267

2285540

928918938017202132290658

12 452

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into heme anid inicreased by about 50 % the rate ofincorporation into heme of radioactive stuccinate.

Pa thzcways of Propionttte FPoriiumtion. Determin-ationi of the rate of conversioni of ' 4C-labeledlactate into heme in whole no(lnlles provi(le(l an

Table IV. Effect of Adding Propionate on the Rate ofIncorporation of Succinate-2-1 4C into Heme

in Soybean NodulesInitact 39 day-old soybean nodules were utilized follow-

ing the conditions of the standard incubation procedure.Five ,uc of succinate-2-'4C were added to all reactionmlixtures and non-radioactive propionate was added as in-dicated. Incubationi was carried out on a slhaker at 26to 280.

Hoursinculbated

3355

0-

o: O

>8E

0.

U 0._a)a

Ln

1000-

800-

600-

400-

200-

Propionateadded

(pmoles)

0

120

12

SI) activityof heme

(cpm/,,umole)

338182440313

1 2 3 4 5 6 7 8

Time of incubation (hr)

FIG. 2. Effect of ad linii acetate on the rate of incor-l)oration of propionate-2-1 4C and succinate-2-'4C illtothe heme moiety of leghemoglobiin in intact soybean nod-ules. Each incubation mixture contained 5 Ac of radio-active metabolite and 12 Mnmoles of unlabeled metabolite.The Standard Incubation Procedure w-as followed witlhthe exception that 12 ,umoles unlabeled acetate were addedas indicated. The nodulles utilized were 39 days old.

inidicationi that propionate mnay be an illterme(liateinl lactate tutilization (table \'). Both lactate-2-14Cand lactate-1-l4C were converte(l into the heme

lmoiety of leghemoglobin at nearly ecllual rates. Theadditioni of unlabeled propioniate to the reactionmixtLure (table V) restulted in a reduiction in thera-lte of 'AC inicorporation into heme from bothlactate-1-_4C (59 %) and lactate-2-'4C (46 %).

\When the remaining propionate was isolated an(l

axssayed, it was apparent that 14C from radioactivelactate was incorporated into propionate. Since the(lutrationi of these experiments was 5 and(I 8 hours,the label cotuld have been coinverted into propioinatevia anl in(lirect route. The data presented in tableII tusing a cell-free extract instead of whole no(duilesshows that the addition of 20 umoles of propionateredutced the rate of incorporation of lactate-1-1+Cinto heme by approximately 30 %. This fuirtherstupports the hypothesis that propionate is an inter-mediate in the conversion of lactate inito heme.

By the tuse of cell-free extracts from niodtulebacteroids, the enzymatic conversion of lactate intopropionate was studied. Data in table VI showthat the conversion of lactate to propi,onate inreaction mixttures containing nodutle extracts re-

qllired ATP, CoA, NADH and( Mg+- as cofactors.The time course for the conversion of lactate-1-14Cinito propionate was fotn(id to be linear for a periodof 30 minuttes (fig 3). The rate of conversioln oflactate-2-14C into propionate was approximatelyequal to the rate of conversion of lactate-1_'4Cinto propionate. These results are in harmony withthose presented in table V. In another experimenitthe adldition of 10 /Lmoles acrylate to an incubationimixtulre identical with the complete reaction mixtuiredescribed in table VI reduced by 54 % the rate offormation of radioactive propionate from lactate-1-14C. These restults are consistent with the possi-bility that acrylate may be an intermediate in thepathway.

The rate of coniversion of radioactive lactateinlto acetate and CO. also was followed in aInexperiment tusing a cell-free extract and the completereaction mixtuire in table V. The loss of radio-activity from lactate-1- 4C as '+CO. was appreciable(330 cpm) after a 20 minulte incuibation period at380 while very little CO., became labeled fromlactate-2-14C (17 cpm). Duirinig this time, the 14C

Table V. Effect of Adding Proprionate on the Rate of Incorporation of Lactate-14C into flcmc in Soybean NodulesNodules xvere inicubated following the procedure outlined in the Materials and Methods xN-ith the exception that

propionate was added as indicated. To each reaction mixtture was added 12 umoles of lactate containing 5 juc of 14C.

Metabolite

Age ofnodules(days)

Hrsincubated

Propionateadded

(umoles)

Sp activityof heme

(cpm/,umole)

8414571289529

Lactate-2-14C 44 5 0Lactate-2-14C 44 5 12Lactate-1-14C 39 8 0Lactate-1-14C 39 8 12

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JACKSON AND EVANS-PROPIONATE AND HE.ME BIOSYNTHIESIS

accuimullatioin in acetate from lactate-2-'4C was muichgreater (1565 cpm per Amole acetate) than thatf I - 1 At- 1 '1 37- 1 , Lfrom lactate- 1-1This differencewas not appare2-14C were conv

Q-0-

0~

L

E

0--

0

Ti

FiG. 3. The r

propionate by a clbean ilodIiles Tbfrom 28 dav-ol(lconitainie(d 96 lumilolextract (14 mg I1Ioni-radioactive la

ATP, anidl MgClincorlporation occI

l)lace of above e)

Table VI1. Cofacby a Cell-freThe complete

1.57 ml of 0.0642 ,uc lactate-1-14C,moles: unlabeledATP, 10; MgCl.,,enzyme extract cc

mixture was incutaction mixture winegative control.

14. (22/ cpm per /Lmole acetate) . Experiments have been conidtucted to test the

in the rate of label accuimulation hypothesis that propionate serves as an alternate

:nt when lactate-1-14C and lactate- carbon source for the supply of succinyl-CoA that

erted into propionate. may be utilized either for heme biosynthesis or for

the maintenance of the citric acid cycle. The reportby De Hertogh et al. (10) that propionate-14C isconverted into intermediates of the citric acid cyclehas been confirmed.

Radioactive tracer experiments have demon-strated that propionate canl be incorporated into theheme componeint of leghemoglobin in intact noduilesor nio(duile extracts (table III, fig 1). It appearsthat propioniate is uitilized withouit a lag period and(that the rate of '4C inicorporation into heme islinear with time. The fact that propilonate-2-14Cnlot only is incorporated into intermedliates of thecitric acidl cycle, but also serves as a precursor ofthe heme moiety of leghemoglobin, is consistenit withthe hypothesis that this compouind makes possiblea secoid(I mechanism for the formation of suiccinyl-CoA. Admittedly the rates of ilncorporationi of 14Clabeled propioniate inito heme are relatively low ullt,they are equlal to or higher than the rates of in-corporationi of several citric acid cycle initermediates.

l l It has been established (10) that cobalt (leficienicy5 10 15 20 25 30 in Rhiwiobiuni miicliloti resuilts in a limited suipplyime of incubation (min) of B,.2 coenzyme and( that this lesionl blocks the

sylnthesis of succinyl CoA from propioniate. The

aell-freeeotractof oactate-dsfi-oC itov possibility is presenited therefore that reduiced leg-- hemoglobin contents of cobalt (leficienlt soybean

e extract was prel)ared froml bacteroids

nlo(11les. The fiiial volulmie of 1.5 nil niodutiles is catused by a failture to efficienitly uitilizeles l)hosl)hate buiffer (pH 7.0); 0.1 niil propioniate. The siginificanice of relative rates ofrotein); 2 uc lactate-l-'4C in 20 /Anioles incorporationi of metabolites into heme is (liffictult toLctate; 10 /Amoles each of prolionate, determinie because of the (lifferenlt sizes of enido-1,; 0.2 /umole CoA anid NADH. No genouis organic acid pools that are present and theirred +-hen boiled extract was used iil lack of information regarding eqtuilibration of in-

Ktract. ternal pools with external additions. No data

obtained proving net synthesis of heme duiring the

incubation periods in either wvhole nodtules or in

for Rcquircents for Lactatc Utilization cell-free extracts of noduiles and the possibility thatc Extract from Nodule Bacteroids '4C accuImuilation in the heme moietv of leghemo-

reaction mixture in a final volume of globin occulrred from exchange reactions mediatedir phosphate buffer pH 7.0 containied by the reversible of the system has not

(5.47 ttc/,.tmole) and the following in been ruled ouit. It established that incorporation

Ilactate, 20: unlabeled propionate, 10 of radioactive propionate and other metabolites into,10; NADH, 0.2; CoA, 0.2 anid 0.1 ml

ofproceeded enzymaticand othe noliesuinto

)ntaining 14 mg protein. Each reactioni heme proceeded enzmatically since no measurable

bated at 360 for 20 min. Complete re- 14C was incorporated when nodules or noduile ex-

ith enz-me extract omitted served as a tracts were boiled.

Enzyme activity

System

Complete-CoA-NADH-mg++-ATP

(total cpm incorporated into10 Amoles propionate in 20 min)

1480210330320400

Since propionate did not occur in nodules indetectable amotunts (table I) it mtust be concludedthat the acid, if present, is utilized as rapidly as itis forme(l. A search was initiated, therefore, toidlentify possible precuirsors. It has been reported(22) that the interior of nodules is nearly anaerobicaud that lactate may be fermented by nodutle bac-teroi(ls (5). This information sutggested that thepathway where lactate is converted to propioniateVia acrylate (3) may be operative. Results pre-

Discussion

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1'1,\N1 1'nPLAN T SIOi,()(A

selitell in tal)les IV and(l V are consistent with thel)ostulation that lactate may serve as a preciurisor- ofpropioniate. Experimenits in which cell-free extractsof l)acteroi(ls were uitilize(l provi(le(l convincingevi(lence for the enizymatic conversioni of lactate topropionate (table VT). The ad(litioni of the possibleinitermediate, acrylate, to the reactioni mixtutre re-(Iticed by approximately 50 % the rate of conversionof lactate-1-11C into propionate, but the possibilitythat acrylate was inhibitory has nlot been ruled out.

The rate of conrversion of lactate-1-1+C intopropionate and into the heme moiety of leghemo-globin is approximately equial to the rate of con-version of lactate-2-14C into these compouinds. Thiswouild niot be expected if the major pathway involvedla conversion of lactate to pyrtivate and then topropionate after decarboxylation. From the resultspresented in table VI it seems highly probable thata major portion of the added lactate is convertedto propionate via a rotute involvinig lactyl CoA andacrylyl CoA as intermediates (3). The conversionof some lactate to pyrtvate is suipporte(d by the factthat '4C from lactate-1-14C an(l lactate-2-14C wasenzymically converted to acetate and CO., uinderanaerobic conditions. MIuich of the label from lac-tate-1-'4C, however, was lost as '+CO., thuis cauisingmutch greater accuimulationi of '"C in acetate fromlactate-2-14C than from lactate-1-14C.

Acknowledgment

The autl-ors express their appreciation to Dr. Te MayChinlg, Dr. WV. W. Clilcote, anid Mr. Sterlinlg A. Russellfor tlheir review of this manuscript.

Literature Cited

1 .AIIME), S. A.xm') H. J. EVANS. 1960. Cobalt: Amicroniutrienit elemenit for the growth of soybeanl)lants utider symbiotic coniditiois. Soil Sci. 90:205-10.

2. A IMED, S. AND H. J. EVA.NS. 1961. The esseni-tialitv of cobalt for soybean plants gro n underSymbiotic conditionis. Proc. Nat]. Acad. Sci. 47:24-36.

3. BALDtIN, R. L., XA. A. NXXooo), AND R. S. E-MERY.1965. Lactate metabolisimi b) Peptostrcptococcusclsidcni: Evidence for lactyl-Coenzvnie A delhv-drase. Biochimii. Biophys. Acta 97: 202-13.

4. PR.NEsS, L. A., D. G. Yo.N(G, AN) R. NOCHO.1963. Methvnlmalonate excretio in vitaimin B 12deficienc\. Sciekice 140: 7677.

5. BITRRIS, R. H. ANI) P. XV. WILSONN. 1939. Res-piratory enzyme s stems isnx bilotic nitrogein fix-altionl. Cold Spring Harbor Svmip. Quanit. Biol.7: 349-61.

(. CH u, T. C. AND E. CHu. 19:55. Paler CliroImdato-graph) of iron complexes of porphyrinis. J. Biol.Cliem. 212: 1-7.

7. COATES, M. E. AND J. E. FORI). 1955. Methods ofmileastirement of vitamiin B12. In The Biocchemis-trv of Vitamini B .. R. T. XVilliams, edl. Lonidon.

(Cambridge( I. IliverSitv 1Press. ( 1IOclebiln. Soc. S \ 1)11.No. 13). 1) 36-51.

8. D)AWx SON, R. M. C., 1). C. Ei.i.io-i W. 11. Et..Lio,i 1.AND K. ,M.. JONES. 1959. Data for- BiochemicalResearch. R. AM. C. DaxNxson1, e(l. Ox ford Uni'Press. p) 284.

9. DIE HERTOGH, A. A. AND H. J. EVANS. 196X2.Studies oIn methmIialonv] isomerase froii Rh1iz_-bium. PlanIt Phvsiol. 37: viI.

10. DE HERTOGH, A. A., P. A. MAYEUX, AN!) H. J.EVANS. 1964. The relationslhip) of cobalt re(q;ire-meInt to prop)ionate metabolism11 in Rhizobliun. J.Biol. Ch1em11. 239: 2446-53.

11. GALL, L. S., S. E. S.MIT11, 1). E. BECKER, C. N.STARK, AN!) J. K. ILOOSLI. 1949. Rmniiieni bacteriain cobalt-deficient shieel). Scienice 109: 468-69.

12. HARTREE, B. E. F. 1955. In Modern Methiods ofPlaInt Analysis. K. Paecli and M. V. TraceV, ec(ls.Berlin: Springer-Verlag, V-ol. IV, 1) 241.

13. HTLCIIER, F. H. AXND XV. VISHNIAC. 1959. Thesite of cvtochrome f lhemiie in spinach chloroplastsati(l somiie of its properties Brookhaven S! mll).Biol. 11: 348-52.

14. KIESE, MI. AND H. K\TRZ. 1954. Untersuichiuingenijiber Cvtochrome. IV. Trenniiunlg vo<n Fer-milenl-thdiimin uinld Protoliimin. Biochlemz. Z. 325: 299312.

15. KL.IEWER, M. AND H. J. Ev.NXS. 1963. Identifica1-tioIn of cobamiii(le coenzyme in nodu(lles of smbl)iontsail(I isolation of the Bi., coenznmefronw iR fwilb1

s/liloti. Planit Physiol. 38: 55-59.1(. LowNE, R. H. AND H. J. EVANS. 1962. Cobalt re-

(luiremenit for the growth of rhizobil. J. Bacteriol.83: 210-11.

17. LOWE, R. H., H. J. EXANS, AND S. AIMNIED. 1960.The effect of cobalt ont the growthi of Rhiomabmio cuin. Biochem. Bioph s. Res. Commun. 3675-78.

18 NICHIOLAS, D. J D., M. KOBAYASIII, ANI) P. XV.XWILSON. 1962. Cobalt relitlireiluenit for inorganicnitrogeni metabolism in microorgalnlils. Prko.NatI. Acad. Sci. 48: 1537-42.

19. ORWMEROD, J. G. 1956. The iuse of ra(lioactiVe car,-)on (lioxi(le in the measniremilenit of carbon (lioxi(lefixation in Rlwdospiri!mini rid(w/i,,l. Biocliem. J.64: 373-80.

20. RIcHMOI(ND, J. E., K. I. AI.TM.AN, AND 1K. SALoXIoN.1954. Fuirther studies oin the biosyntlhesis of heminin bone marrowx l)relparations. J. Biol. Chemll. 211:981-88.

21. RICHMOND, J. E. AND K. SALOMNIoN. 1955. Stut(liesoni the biosynthlesis of lhem1ini in soybeanneodIilles.Biochemii. Biophys. Acta 17: 48-55.

22. S-mc1n!i, J. D. 1949. Thie coilcenitratioli and dhistr1iblution of lhemiioglobin in the root 1lo(hI!les of legni-1111)o01s lplants. Bioclhemii. J. 44: 585-91.

23. Swim, H. E. AND MI. F. UTTrER. 1957. Isotopicexplerimelntation Nv itlh intermediates of the tricar-boxvl ic aci(l c cle. In: Methods in Enzvmology.S. P. Colowx ick and N. 0. IKaplan, e(l. Vol. IV.Academiiic Press, I!lc. Newx York. 1) 589-609.

24. TING, I. P. AND Wv. M. DUGGER, JR. 1965. Separa-tioII anld detection of organic acids oni silica gel.Anal. Biochem. 12: 571-78.

25. XXLT.sON, D. 0. AND H. MI. REISENAUER. 1963. Co-halt re(quirem-lenit of sv lliotically grown al fal fa.Plant Soil 19: 364-73.

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Propionate in Heme Biosynthesis in Soybean - Plant Physiology