Vol. 64 NUCLEAR ALDOLASE 373lated nuclei. The author has observed that 90-95%of the total nuclear aldolase could be extracted with015M-NaCl, and Kirkham & Thomas (1953) havereported a globulin fraction readily extracted with0. 14m-NaCl from nuclei isolated in organic solvents,the fraction comprising 30-50% of the dry weightof the nuclei.

Until more is known of the nature of the bindingbetween aldolase and nuclei, and until proteinredistribution can be prevented (at least after thehomogenization process) by the use of betterisolation media, it cannot be categorically statedthat aldolase is a true component of liver nuclei.

SUMMARY

1. A study has been made of factors affecting thealdolase activity of rat-liver nuclei.

2. The method of homogenization affectednuclear aldolase. Treatment ofthe homogenate witha Waring Blendor for 30 sec. resulted in a 66% fallin the total enzyme recovered in the nuclearfraction.

3. Delay in isolating the nuclei after the pre-paration of the homogenate resulted in alterationsin nuclear activity. In one fractionation a delay of12 min. resulted in the loss of 47 % of the nuclear-enzyme. The fall in activity was coincident with arise in pH of the homogenate.

4. Isolation of nuclei from weakly bufferedhomogenates showed that the aldolase activity ofthe nuclear fraction was greatly affected by the pHof the homogenate, a nuclear fraction obtained atpH 6-0 having 3X3 times the activity of one isolatedat pH 6-75.

5. Alterations in nuclear activity were due to a

redistribution of enzyme between nuclei and super-natants and not to activation or inactivation.

6. Indirect evidence suggested that the effectsobserved with aldolase applied to other nitrogen-containing materials.

7. It is concluded that the aldolase activity ofisolated nuclei is greatly affected by the method ofisolation of the nuclei.The author would like to thank Dr M. Dixon, F.R.S., and

Dr E. C. Webb for helpful advice and criticism, and also theMedical Research Council for a research grant given duringthe course of this work.

REFERENCESAllfrey, V., Stern, H., Mirsky, A. E. & Saetren, H. (1952).

J. gen. Phy8iol. 35, 529, 559.Dounce, A. L., Witter, R. F., Monty, K. J., Pate, S. &

Cottone, M. A. (1955). J. biophys. biochem. Cytol. 1, 139.Duve, C. de & Berthet, J. (1954). Int. Rev. Cytol. III,

p. 225. New York: Academic Press.Duve, C. de, Pressman, B. C., Gianetto, R., Wattiaux, R. &Appelmans, F. (1955). Biochem. J. 60, 604.

Hogeboom, G. H. & Schneider, W. C. (1952). J. biol. Chem.197, 611.

Hogeboom, G. H., Schneider, W. C. & Striebich, M. J.(1952). J. biol. Chem. 196, 111.

Kay, E. R. M., Smellie, R. M. S., Humphrey, G. F. &Davidson, J. N. (1956). Biochem. J. 62, 160.

Kirkham, W. R. & Thomas, L. E. (1953). J. biol. Chem. 200,53.

Maver, M. E., Greco, A. E., Lovtrup, E. & Dalton, A. J.(1952). J. nat. Cancer In8t. 13, 687.

Palade, G. E. (1951). Arch. Biochem. 30, 144.Philpot, J. St L. & Stanier, J. E. (1956). Biochem. J. 63,214.Roodyn, D. B. (1956). Biochem. J. 64, 361.Stern, H. & Mirsky, A. E. (1953). J. gen. Phy&iol. 37, 177.Wilbur, K. M. & Anderson, N. G. (1951). Exp. Cell Re8. 2,

47.

The Use of Radioactive Carbon Dioxide in the Measurementof Carbon Dioxide Fixation in Rhodospirillum rubrum

BY J. G. ORMEROD*Department of Microbiology, The Univer8ity, Sheffield

(Received 14 March 1956)

The Athiorhodaceae utilize a wide variety oforganic compounds for growth. Light is essentialfor anaerobic growth, but most of these organismswill grow in the dark in the presence of oxygen.Growth in the light is dependent on chlorophyll.According to the hypothesis ofvan Niel (1941, 1944,1949) the primary light reaction in the photo-synthetic bacteria is the same as that in green plant

photosynthesis and involves the splitting of waterwith the formation of a reduced component and anoxidized component. The former is thought toreduce carbon dioxide, presumably in the form of1:3-diphosphoglyceric acid (Bassham et al. 1954),whereas the oxidized component is the terminalacceptor in the oxidation of the substrate. In thelight most of the organic substrate is assimilated aswas shown by van Niel (1944). Gaffron (1933, 1935)was the first to use manometric methods for the

* Present address: Department of Biochemistry, TheTechnical University of Norway, Trondheim, Norway.

J. G. ORMEROD

study ofthe photometabolism oforganic compoundsby washed suspensions of these organisms. Hefound that with substrates more reduced than cellmaterial there was a fixation of carbon dioxide, theamount fixed depending on the state of reduction ofthe substrate. In substrates more oxidized than cellmaterial there was an output of carbon dioxide.Van Niel (1944) has made similar observations.

It is more than likely that carbon dioxide pro-

duction and carbon dioxide assimilation go on

simultaneously in the light, and this means that it isimpossible, with only manometric methods, toobtain figures for the absolute amount of carbondioxide produced or assimilated; the best one can

hope for with this method is figures for net produc-tion or net assimilation. 14C-Labelled carbondioxide offers a possible solution to this difficulty,and Cutinelli, Ehrensvard, Reio, Saluste & Stjern-holm (1951) have shown that when Rhodospirillumrubrum is grown in the light upon acetate and 14CO2some 10% of the cell carbon is derived from thecarbon dioxide. Even this method is not withoutobjection, for exchange reactions involving carbondioxide may well occur without any increase in thetotal carbon of the system.For an understanding of the part played by

carbon dioxide in the photometabolism of theseorganisms it seemed worth while to attempt tomeasure the amount of carbon dioxide fixed with14CO2, and to compare the results with those ob-tained by the manometric method.

MATERIALS AND METHODS

Organism and cultural procedure

The organism used was Rhodpirlum rubrum strain SI,from the collection of Dr C. B. van Niel, Hopkins MarineStation, Pacific Grove, California, U.S.A.The maintenance of the organism and the preparation of

inocula have already been described (Elsden & Ormerod,1956). For use in experiments the organism was grown

on a DL-malate-(NH4)2SO4-yeast extract-salts medium(Elsden & Ormerod, 1956) and harvested after growth for40-45 hr. at 250 in the light cabinet. Cells grown on a similarmedium containing 0-4% (w/v) of sodium DL-lactate and0-3% (w/v) ofNaHCO3, or on 5% (v/v) of yeast autolysate(Barker & Beck, 1942), were used for experiments involvingDL-lactate or propionate as substrates.

Preparation of washed suspensions. Washed suspensionscontaining 5 mg. dry wt. of cells/ml. in 0-0049m-NaHCO3were prepared as previously described (Elsden & Ormerod,1956).

Substrates

All organic acids were used as solutions of their sodiumsalts. The succinic acid, DL-lactic acid, and sodium acetatewere A.R. grade. Crystalline sodium pyruvate was pre-

pared from crystalline pyruvic acid by the method ofRobertson (1942). The propionic and butyric acids were

purified by distillation (b.p.'s 1410 and 1610 respectively).

Sodium L-malate and fumarate were prepared from thecommercial acids obtained from Light and Co., Colnbrook,Bucks. A solution of radioactive Na2CO, was prepared fromBa14CO3 obtained from the Radiochemical Centre, Amer-sham, Bucks, and diluted with inactive carrier Na2CO3 asrequired.

General analytical methodsVolatile fatty acids. These were estimated by steam-

distillation in the Markham still (Markham, 1942) andtitration of the distillate with C02-free 0-01 N-NaOH in astream of C02-free air.

Pyruvic acid. This was estimated by the carboxylasemethod ofWesterkamp (1933); the carboxylase preparationwas stable for at least 3 months if stored at - 200.

Lactic acid. This was determined by the ceric sulphatemethod of Elsden & Gibson (1954).

Methods used with radioactive materials

Preparation and assay of radioactive samples. Singleorganic compounds were assayed for 14C activity afteroxidation to CO2 with the combustion mixture ofVan Slyke& Folch (1940). The CO2was absorbed in CO-free 3x-NaOHand precipitated as BaCO3 by the method described below,which is a modification of that published by HenriquesKistiakowsky, Margnetti & Schneider (1946). The modifica-tion is due to the Radiation Committee of Western ReserveUniversity, Cleveland, Ohio, U.S.A., and was introducedinto this laboratory by Dr H. J. Saz. The filtration assemblywas essentially that described by Henriques et al. (1946).A tared filter paper (Whatman no. 40) was placed on thesintered-glass disk and the stainless-steel collar fitted overthis and held in position by two springs. An excess (approx.2 ml.) of CO2-free 5% (w/v) BaCl2 solution was run into thewell over the filter paper, and the sample solution containingcarbonate equivalent to 10-15 mg. of BaCO3, diluted, ifnecessary, with inactive carrier (01OM-Na2CO.), was addedslowly from a pipette. The pH of the solution was alwaysgreater than 10. The precipitate of BaCO3 was filtered on tothe paper by suction, and formed a uniform circular deposit19 mm. in diameter. This was washed successively with CO2-free water, 50% (v/v) aqueous acetone and acetone. Thepaper was then removed from the filtration assembly anddried by placing under a 250w infrared lamp at a distance of22 in. for 1-5 min. The paper was then left at room temp. for1-2 hr. to equilibrate with the moisture ofthe air, reweighedand the weight of BaCO3 calculated. The paper disk bearingthe sample was then mounted on a brass block (Henriqueset al. 1946) and the radioactivity measured with a Geiger-Mfuller end-window counter. The activity was corrected forbackground and self absorption and the radioactivity isexpressed as counts/min. at infinite thinness. Stock solu-tions of Na214CO2 were assayed by the same method.The total radioactivity of bacterial suspensions was

measured directly (Calvin, Heidelberger, Reid, Tolbert &Yankwich, 1949) by delivering 0-05 ml. of the well-mixed,acidified suspensions on brass disks from an Agla micro-meter syringe. (Burroughs, Wellcome and Co., London).The disks measured 25-0 mm. in diameter and were 1-3 mm.thick. A circular groove of internal diameter 19-0 mm. and0-25 mm. deep was cut into the surface of each disk. Thesuspension on the disk was neutralized by adding therequired amount of C02-free 0-01 N-NaOH from a micro-meter syringe and the mixture spread evenly over the entire

I956374

CARBON DIOXIDE FIXATION IN RHODOSPIRILLUMsurface of the disk within the groove with a bent nichromewire, cleaned before use by flaming. It was found that theprocedure used for decontaminating the disks (5-10 min. inchromic acid cleaning fluid followed by thorough washingwith water and drying on filter paper) ensured even spreadingof the suspension. The disks were then placed under a250w infrared lamp at a distance of 12 in. to dry; the timerequired for complete drying was about 7 min.The same geometry was used when counting these

samples as when counting BaCO3. With this technique, thedried layer of radioactive material was about 0-2 mg./cm.2thick. When counting such thin layers the corrections forself-absorption are small by comparison with other errors inthe procedure, and can be neglected (cf. Calvin et al. 1949).The values obtained by this method compared favourably-with those obtained by oxidizing the cell suspension asdescribed above and counting as BaCO3 (Table 1). Thestandard error of the counts in Tables 2-4 was, in themajority of cases, less than 3 %, and in no case greater than5%.

Isolation of propionic acid. The acidified suspension wascentrifuged and the cells were discarded. A sample of thesupernatant containing about 30 ,imoles of propionate wassteam-distilled in the Markham apparatus and the distillatetitrated with CO2-free 0.01 N-NaOH in a stream of C02-freeair. Propionic acid was separated from the distillate bychromatography on Celite buffered with 2M phosphate atpH 6-5 (Bueding & Yale, 1951). The column was developed

Table 1. Comparison of racdioactivity of cellsuspensions with different assay procedures

Radioactive cell material was obtained in duplicateexperiments by incubating cell suspensions in the light inthe presence of 14CO2, with and without substrate. Cupswith double-side bulbs contained in the main compartment2-0 ml. of washed suspension of cells (5 mg. dry wt./ml.)in 0-0049M-NaHCO3, and 0-8 ml. of water. The first sidebulb contained 0-05 ml. of 0-483M-Na214CO3 (220 x 103counts/min.) together with 0-16 ml. of water, and thesecond contained 0-2 ml. of 0-05M substrate solution(fumarate or L-malate). After gassing with 02-free N2,0-04 ml. of ION-H2SO4 was injected into the carbonatesolution in the first side bulb and after 30 min. equilibra-tion in the dark, the substrate was tipped and the lightswere switched on. When all the substrate had disappeared,acid from the side bulb was tipped in cups both with andwithout substrate, and the concentrations of NaHCO3were measured. The manometers were then flushed toremove residual CO2 and the radioactivity of the cellsuspensions was assayed both by oxidation to CO2 and as

BaCO3, and by direct plating on brass discs.

Counts/min./manometer cupA_-_ Duration

Counted Counted direct of expt.Substrate as BaCO3 after drying (min.)L-Malate 66 500+710 67 500+670 265

67 200±720 67 000+666Fumarate 42 560+±322 42 900+542 191

43 720+322 42400+534None 24 200+226 24 300+410 191

24520+230 24 750+41024 600+410

by the addition of CHC13 containing increasing concentra-tions of n-butanol, and the fractions were titrated in astream of C02-free air with C02-free 0-01 N-NaOH. Thefractions containing propionate, which was the only acidpresent in detectable amounts, were pooled and the aqueouslayer containing the propionate was separated and steam-distilled. The distillate was titrated, evaporated to drynessand oxidized, and the C02 converted into BaCO3 and countedas described above.

Isolation and degradation of sUCCinic acid. A portion of theacidified suspension was centrifuged and 1-0 ml. of thesupernatant, containing approximately 10 ,umoles ofsuccinic acid, was extracted with ether for 2 hr. in aKutscher-Steudel extractor. To the ether extract was added1-0 ml. ofwater, and the ether was removed by evaporation.The pH of the aqueous extract was adjusted to less than2 with 2N-H2SO4, and separation of succinic acid from thissolution was carried out by chromatography on a Celitecolumn (Swim & Krampitz, 1954). The fractions containingsuccinate were pooled and the aqueous layer was separatedand evaporated to dryness. The residue was made up to3 0 ml. with water, and 1-0 ml. of this solution was driedand the product oxidized; the C02 so obtained was con-verted into BaCO3 and counted.A second portion of the succinate solution was converted

into propionate and C02 by a suspension of Veillonellagazogenas (Swim & Krampitz, 1954). The strain used wasisolated from saliva by the procedure of Johns (1951 a). Thedecarboxylation was carried out under the conditionsspecified by Johns (1951b) in a Warburg manometer witha double side-bulb cup, one side bulb ofwhich was fitted witha gassing vent to facilitate removal of the C02 produced bydecarboxylation. The main compartment of the manometervessel contained the sample to be decarboxylated (1.0 ml.),together with 1-75 ml. of 0- 1 M potassium phosphate buffer,pH 5-8. The bacterial suspension (0.5 ml.) was placed in oneside bulb and 0-2 ml. of 2N-H2S04 in the other. A controlcup was run containing 1 0 ml. ofwater in place of succinate.The manometers were gassed with 02-free N2. When thereaction was complete the manometer was removed fromthe bath and an absorption vessel containing C02-free3 N-NaOH was connected to the vented stopper. The mano-meter was flushed with C02-free air for 35 min., whichexperience had shown was sufficient to remove all the C02.The C02, which had been trapped in 3N-NaOH, was con-verted into BaCO3 and counted. The acidified suspension wasthen removed from the manometer and the propionate, theother product of the decarboxylation, separated by steam-distillation. The distillate was titrated with C02-free0-01 N-NaOH, evaporated, and the residue so obtainedoxidized and the C02 converted into BaCO3 and counted.Since succinate behaves as a symmetrical molecule in thissystem it is possible to measure the radioactivity of thesuccinate carboxyl groups. The activity in the methylenecarbons of the succinate is estimated by difference.Manometric procedure. Gaseous exchange was measured

in Warburg manometers which had stopcocks fitted to theleft-hand limb. Cups of special design with double sidebulbs were used. The first side bulb was provided with a Blosocket which was closed with a Blo cone to which was fuseda capillary stopcock (1 mm. bore). The second side bulb hadan outlet into which could be fitted a turnover-type rubberstopper (Wm. Freeman and Co. Ltd., Subaseal Works,Barnsley, Yorks). Substrates (0.2 ml.) were placed in the

Vol. 64 375

J. G. ORMERODfirst side bulb and 0 16 ml. of water was placed in the secondtogether with 0 05 ml. of Na214CO2 solution, added from amicrometer syringe. The rubber stopper was then fixed intoplace. The total volume of bacterial suspension plus sub-strate was 3 0 ml., and the concentration of bicarbonate was0-00326 M as measured by acidification in a separate mano-meter. The concentration of the Na214CO3 solution used wasdetermined in a similar manner and found to be 0-483M.Thus the initial CO2 content of the manometers was33-9 ,umoles. The Warburg bath used was that described byElsden & Ormerod (1956). The manometers were placed inthe bath (temp. 300), gassed with 02-free N2 for 4 min., andafter equilibration the levels of the manometric fluid wereset high in the right-hand limb and low in the left. Thestopcock on the left-hand limb of the manometer was closedto prevent movement of the manometric fluid, the mano-meters were removed in turn from the bath and 0 04 ml. ofION-H2SO4 was injected into the side bulb containing theradioactive Na2CO., through the rubber stopper with amicrometer syringe. This amount of acid was sufficient forboth liberation of CO2 from the carbonate solution andacidification of the cell suspension at the end of the experi-ment. The manometers were then replaced in the bath andshaken in the dark until all pressure changes had ceased(30 min.). The stopcock on the left-hand limb of the mano-meter was opened and the level ofthe manometric fluid in theleft-hand limb was then suitable for measuring gas uptake.The substrate was tipped and the lights were turned on.Parallel manometers to record the endogenous metabolismwere set up in the same manner but with the substratesolution replaced by water.

Disappearance of substrate was followed by measuringpressure changes, and when all the substrate had beenmetabolized (indicated by a sharp break in the C02-uptakecurve), the excess of acid was added from the side bulb andthe final bicarbonate concentration measured. The finalbicarbonate concentration in the cup without substrate wasmeasured at the same time. In certain cases the reaction wasstopped before all the substrate had been utilized, and theconcentration of the substrate remaining was estimated.When equilibrium was again reached (10-15 min.) thestopcock on the left-hand limb of the manometer was closedand the manometers were removed from the bath unopened.An absorption vessel containing 4-0 ml. of C02-free 3w-NaOH was connected to the stopcock on the side arm. Themanometer was then flushed for 35-40 min. with C02-freeair, with frequent shaking during the flushing period. TheCO2 trap was then removed and the manometers wereflushed with N2 + C02 (95: 5) to wash out the last traces of14CO2. The alkaline solution containing the CO2 from the gasphase was diluted with water and 0O10m-Na2CO, solution,converted into BaCO2, and counted. The radioactivity ofthe cell suspensions was measured directly as describedabove.

Calculation of results from manometric and isotope data.The pressure changes occurring when the sodium salt of anorganic acid is metabolized in the light by suspensions ofR8p. rubrum in bicarbonate-CO2 buffer are the result ofthree processes: (a) binding of CO2 by the buffer due toremoval of anions (the increase in bicarbonate resultingfrom this process can be measured by acidification);(b) fixing of C02 by photosynthesis; (c) production of CO2 asa result of oxidation of the substrate; since with many sub-strates there is a simultaneous output and fixation of C02,

the manometric method provides a measure of only the netchanges. If the increase in the bicarbonate is less than theC02 uptake observed during the metabolism of the sub-strate, the difference between the two values represents anet fixation of CO2, whilst if the bicarbonate change isgreater than the observed CO2 uptake, the differencerepresents a net CO2 output.

In the procedure used in the present experiments, cellsuspensions were incubated in the dark for 30 min. beforethe lights were switched on. During this equilibrationperiod some endogenous fermentation occurred and, whenthe lights were switched on, the fermentation products thathad accumulated were metabolized with an uptake of CO2which usually amounted to 10-30,ul. The amount of 14CO2which was incorporated in this way never correspondedexactly with the manometric results and was generally muchhigher. Withisotopedata, the quantityof14CO2incorporatedis calculated from radioactivity measurements made oncells incubated with and without substrate. Thus:

,umoles of CO2 incorporated =(counts/min. fixed in cells with substrate - counts/min.

fixed endogenously) x initial CO2 (Qmoles)counts/min. in initial C02

It is assumed that the deduction of the endogenous values islegitimate. In what follows the terms net fixation and netoutput of CO2 refer to the results obtained by manometricmethods. Incorporation of 14CO2 is defined as the amount ofCO2 (expressed as ,umoles) taken up by the cells, as measuredby the tracer technique, whether the CO2 entered thesystem by exchange reactions, by fixation reactions or bya combination of the two.

RESULTS

Incorporation of 14CO2 with acetate, propionate andbutyrate as substrates. The values obtained for 14CO2incorporation and for net C02 fixation or net CO2output are presented in Table 2. Only with butyratedid the figure obtained by the isotope methodcoincide with that for net C02 fixation. With pro-pionate the amount of 14CO2 incorporated was morethan twice as great as the net fixation. In theacetate experiments the number of counts fixedin the absence ofsubstrate was in all cases except onesignificantly greater than when acetate was present.The manometric results obtained with acetate showthat there was a net C02 output of 0 2-0 3 ,umole/,mole of acetate metabolized, a value similar tothat obtained by van Niel (1941).

Pyruvate and DL-lactate as substrates. Table 3shows the result obtained with these substrates for14CO2 incorporation. In addition, the manometricdata are given. When pyruvate was the substrate,the net output of C02 was large, and the amount of14CO2 incorporated was small by comparison. Onthe other hand, substantial quantities of 14CO2 wereincorporated with lactate, a substrate whose stateof oxidation differs from that of pyruvate by onlytwo hydrogen atoms.

376 I956

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Succinate, fumarate and L-malate a8 8ub8trates.The values obtained with these substrates for 14CO2incorporation and for CO2 output are given inTable 4. With succinate the amount of C02 in-corporated was 0-5 manole/,umole of succinate used.The results for fumarate were very similar to thoseobtained with L-malate. In each case the 14CO2incorporation amounted to 0-28-0-29 umole/pmoleof substrate used, and manometrically there was anet output amounting to 1 1-1 2 pmole/pmole ofsubstrate utilized.

Photometaboli8m of propionate and 8umcinate. Inrecent years an enzyme system has been describedwhich catalyses the decarboxylation of succinate togive propionate. The presence of this system hasbeen demonstrated in Propionibacterium pento-saceum (Delwiche, 1948) and in Veillonella gazogenes(Johns, 1951 b). Reversibility ofthe reactions in thelatter organism has been demonstrated by Whiteley(1953). Larsen (1951) has obtained evidence for theoccurrence in Chlorobium thiosulphatophilum of anenzyme system catalysing the carboxylation ofpropionate to succinate; this reaction is light-dependent. It appears, therefore, that a closemetabolic relationship exists between succinate andpropionate in these organisms. Table 2 shows thatwith propionate the incorporation of 14CO2 is con-siderably higher than the net C02 fixation. SinceChl. thiosulphatophilum carboxylates propionate tosuccinate, it was thought possible that the photo-metabolism of propionate by R8p. rubrum mightalso involve carboxylation to succinate as an initialstep. As succinate is a symmetrical molecule, areversal of the reaction in the presence of 14CO2would result in labelling of the carboxyl group ofpropionate. If added succinate participates in a

similar reaction during metabolism in the light, italso would become labelled in the carboxyl groups,always provided that intracellular substrate is inequilibrium with extracellular substrate (cf. Saz &Krampitz, 1954). To test this point, experimentswere performed in which propionate or succinatewas incubated in the light with suspensions of R8p.rubrum in the presence of 14CO2, and when 15-20%of the substrate had been metabolized the reactionwas stopped, the residual substrate isolated anddegraded and the products were assayed for radio-activity. The results are shown in Table 5. It will beseen that the incorporation of isotope into succinateis sufficient to account for only 2-8% of the totalfixation due to succinate. The incorporation ofisotope into propionate is hardly significant. Forthe experiment with propionate (Table 5) the cellswere grown in 5% (v/v) of yeast autolysate. Theincorporation of C02 as measured by the isotopemethod was 1 2 ,moles/,umole of propionate meta-bolized.

DISCUSSION

The results obtained with acetate suggest that thissubstrate suppresses 14CO2 incorporation. A similarresult was obtained by Glover & Kamen (1951.),who incubated suspensions of R8p. rubrum in thelight with and without acetate, under hydrogen andin the presence of 14CO2. Under these conditions,the quantity of isotope incorporated in the presenceof acetate was less than that incorporated in itsabsence. At present there is no explanation forthese results. It appears that cells of Rsp. rubrumincorporate carbon dioxide into cell material whengrown on acetate (Cutinelli et al. 1951; van Niel,1949).

Table 5. Incorporation of 14C02 into 8uccinate and propionateManometer cups were set up as described under Table 2. The substrate solution contained 40 umoles of succinate or

60 ,umoles of propionate. The reaction was carried out as described in Table 1 and stopped by tipping in acid after approxi-mately 15-20% of the substrate had been metabolized. After determination of the radioactivity of the cell suspension theisolation and degradation of the residual substrates were carried out as described under Materials and Methods. Allradioactive samples were counted to within 2% error, except where stated otherwise.

Succinate expt. (110 min.)

Endogenous With succinateSubstrate used (,umoles)Initial C00 (counts/min.)Initial C00 (,umoles)Total counts fixed in cellsFinal C02 (counts/min.)Sum of radioactivity in final C02 and in cells(counts/min.)

224 00033.9

15200208 000223 200

7224 000

33.938 700184 000222 700

Propionate expt. (150 min.)

Endogenous With propionate

224 00033.9

11 500213 000224 500

9224 000

33.982 000142 000224 000

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SuccinateCounts/min./,umole =93+ 4Counts/min./,&mole in C02H group =41 i 1Counts/min./pmole in propionate formed

in succinate degradation =45+t3

PropionateCounts/min./,umole = 11 +5

378 I956

CARBON DIOXIDE FIXATION IN RHODOSPIRILLUM

With propionate and butyrate as substratesthere was a net fixation of carbon dioxide which canbe measured manometrically, and the result servesas a check on the figures obtained with 14CO2 Withpropionate the net fixation was much less than theamount of 14CO2 incorporated, but at the same timeno isotope was found in the residual substrate. Atfirst sight this suggests that propionate as such isnot involved in a reversible carboxylation reaction;Larsen's (1951) results indicate that in Chl. thio-sulphatophilum this reaction is not reversible sincethe succinate formed from propionate accumulates.However, as Saz & Krampitz (1954) have shown,experiments of the 'carrier' type must be inter-preted with caution since the substrate outside thecell may not be in equilibrium with that inside thecell.

Butyrate was the only substrate with which thenet carbon dioxide fixation agreed with the valueobtained by the isotope method. This result suggeststhat no carbon dioxide is produced during thephotometabolism of butyrate. Nothing else isknown concerning the mechanism ofthe light oxida-tion and assimilation of this substrate.The results obtained with pyruvate as substrate,

namely, a small incorporation and comparativelylarge output of carbon dioxide, are as expected inview of the fact that pyruvate is considerably moreoxidized than cell material. On the other hand, withlactate, which is at about the same state ofoxidationas cell material, the amount of 14CO2 incorporated isquite large. The manometric results with lactateshow that there is a small net output of carbondioxide. It is possible, therefore, that the incorpora-tion of 14CO2 is due mainly to an exchange reactionand does not represent a fixation of carbon dioxide.The quantity of 14CO2 incorporated when succinateis the substrate is equivalent to about 0 5 pmole ofcarbon dioxide/,mole of succinate used. This isin keeping with the scheme for the metabolism ofsuccinate by R8p. rubrum put forward by Elsden &Ormerod (1956) on the basis ofresults obtained withfluoroacetate. In this hypothetical scheme, succi-nate is oxidized to phosphoenol pyruvate by apathway involving fumarate, malate and oxalo-acetate. During this oxidation, two pairs of hydro-gen atoms and one molecule of carbon dioxide areproduced. Subsequent reduction of the phos-phoenol pyruvate to triose phosphate uses one pairof hydrogen atoms and the remaining pair isutilized in the photosynthetic reduction of half amolecule of carbon dioxide. Thus for each moleculeof succinate metabolized, half a molecule of carbondioxide is fixed and one molecule of carbon dioxideis produced.Fumarate and malate, like lactate, gave higher

figures for 14CO2 incorporation than expected on thebasis of their state of oxidation. In connexion with

the results obtained with these three substrates,the experiments of Wood, Vennesland & Evans(1945) are interesting. These workers reported that13CO2 was incorporated into the carboxyl groups offumarate, malate, lactate and pyruvate when thesecompounds were incubated with crude pigeon-liverextracts. The incorporation into malate andpyruvate has since been explained by the discoveryin pigeon liver of the 'malic enzyme' (Ochoa,Mehler & Kornberg, 1947) and the incorporation ofisotope into the other acids was due to the presenceof lactic dehydrogenase and fumarase. Since all ofthese enzymes are present in R8p. rubrum (Vernon &Kamen, 1953; Eisenberg, 1953) it is possible tointerpret in a similar manner the results obtainedwith lactate, fumarate and malate in the bacterialsystem. If the labelled malate formed in thecarboxylation reaction is in equilibrium with asymmetrical compound (e.g. fumarate) a reversal ofthe reaction would give rise to some labelled C3compound. On subsequent assimilation of this,isotope would be incorporated into the cells. Thevalues obtained for 14CO2 incorporation with thesesubstrates may not therefore represent the truegain in carbon.

It is difficult to interpret the result of the experi-ment with succinate, in which a small amount of14CO2 was incorporated into the residual substrate.In the presence of 14CO2, such incorporation couldresult from the action of the 'malic enzyme' onmalate reversibly formed from succinate.

It appears from the results discussed thatmethods involving labelled carbon dioxide areinadequate for measuring carbon dioxide fixation ina photosynthetic system of the type found in R8p.rubrum. The metabolism of organic substrateswhich occurs causes difficulties as certain com-pounds seem to inhibit or be substrates for ex-change reactions involving carbon dioxide. In suchcases it is impossible to estimate by the isotopemethod the quantities of carbon dioxide fixed.

SUMMARY

1. In experiments with suspensions of Rhodo-&pirillum rubrum illuminated in the presence of14CO2 and various substrates, the incorporation ofradioactivity and the net fixation or output ofcarbon dioxide have been measured.

2. In the presence of pyruvate, DL-lactate,succinate, fumarate and L-malate there was a netoutput of carbon dioxide, and with propionate andbutyrate there was a net fixation ofthe gas. Each ofthese substrates caused an increase in the incorpora-tion of 14CO2 over and above that found in thecontrol without substrate. Acetate, which gave anet output of carbon dioxide, depressed endogenousincorporation of 14CO2.

379Vol. 64

380 J. G. ORMEROD I9563. When a suspension of R8p. rubrum was incu-

bated in the light with an excess ofpropionate in thepresence of 14CO2 the residual propionate containedno significant radioactivity.

4. A similar experiment with succinate showedthat the residual substrate contained radioactivitysufficient to account for only 258% of the totalincorporation due to succinate.

5. The implication of these results is discussed.I would like to thank Dr S. R. Elsden for his encourage-

ment and interest in this work, Dr H. J. Saz for his advice onthe use and assay of 14C, and the Agricultural ResearchCouncil for the receipt of a Research Studentship. Thisworkwas supported in part by a grant from the RockefellerFoundation.

REFERENCESBarker, H. A. & Beck, J. V. (1942). J. Bact. 43, 291.Bassham, J. A., Benson, A. A., Kay, L. D., Harris, A. Z.,

Wilson, A. T. & Calvin, M. (1954). J. Amer. chem. Soc.76, 1760.

Bueding, E. & Yale, H. W. (1951). J. biol. Chem. 193, 411.Calvin, M., Heidelberger, C., Reid, J. C., Tolbert, B. M. &

Yankwich, P. E. (1949). In Isotopic Carbon. New York:Wiley.

Cutinelli, C., Ehrensvard, G., Reio, L., Saluste, E. &Stjernholm, R. (1951). Ark. Kemi, 3, 315.

Delwiche, E. A. (1948). J. Bact. 56, 811.

Eisenberg, M. A. (1953). J. biol. Chem. 203, 815.Elsden, S. R. & Gibson, Q. H. (1954). Biochem. J. 58, 154.Elsden, S. R. & Ormerod, J. G. (1956). Biochem. J. 63, 691.Gaffron, H. (1933). Biochem. Z. 260, 1.Gaffron, H. (1935). Biochem. Z. 275, 301.Glover, J. & Kamen, M. D. (1951). Fed. Proc. 10, 190.Henriques, F. C., Kistiakowsky, G. B., Margnetti, C. &

Schneider, W. G. (1946). Industr. Engng Chem. (Anal.),18, 349.

Johns, A. T. (1951a). J. gen. Microbiol. 5, 317.Johns, A. T. (1951 b). J. gen. Microbiol. 5, 326.Larsen, H. (1951). J. biol. Chem. 193, 167.Markham, R. (1942). Biochem. J. 36, 790.Ochoa, S., Mehler, A. & Kornberg, A. (1947). J. biol. Chem.

167, 871.Robertson, W. B. (1942). Science, 96, 93.Saz, H. J. & Krampitz, L. 0. (1954). J. Bact. 67, 409.Swim, H. E. & Krampitz, L. 0. (1954). J. Bact. 67, 426.van Niel, C. B. (1941). Advanc. Enzymol. 1, 263.van Niel, C. B. (1944). Bact. Rev. 8, 1.van Niel, C. B. (1949). In Photosynthesis in Plants, ed. by

Franck, J. & Loomis,E.W. Ames: IowaStateCollege Press.Van Slyke, D. D. & Folch, J. (1940). J. biol. Chem. 136,509.Vernon, L. P. & Kamen, M. D. (1953). Arch. Biochem.

Biophys. 44, 298.Westerkamp, H. (1933). Biochem. Z. 263, 239.Whiteley, H. R. (1953). J. Amer. chem. Soc. 75, 1518.Wood, H. G., Vennesland, B. & Evans, E. A. (1945).

J. biol. C7hem. 159, 153.

The Biosynthesis of Penicillin4. THE SYNTHESIS OF BENZYLPENICILLIN BY WASHED MYCELIUM

OF PENICILLIUM CHRYSOGENUM*

BY W. J. HALLIDAYt AND H. R. V. ARNSTEINNational In8titute for Medical Research, Mill Hill, London, N.W. 7

(Received 30 April 1956)

Although washed mycelial suspensions of Penicil-lium chry8ogenum have been employed for the studyof various metabolic properties of the mould, therehave been few reports on the synthesis of penicillinby such preparations. Penicillin production byreplacement cultures of Penicillium notatum wasbriefly reported by Abraham et al. (1941), whoobserved that maximum yields of penicillin wereobtained more quickly when the original cultureliquid was replaced by fresh nutrient medium.More recently Rolinson (1954) showed that P.chry8ogenum could continue to produce penicillin ata maximum rate when the mycelium was washedand suspended in a fresh, nutrient medium.Rolinson's investigations indicated the possibilityof performing short-term 'fermentations', whichwould yield penicillin in measurable quantities and

be relatively free from the metabolic by-productsof complete fermentations. We were thereforeprompted to examine the effect of various nutrientsand inhibitors on penicillin production by washedmycelium, with a twofold purpose: first, to seewhether washed mycelium forms penicillin from thesame precursors as does the mould under more usualconditions; secondly, to attempt the isolation andidentification of the later intermediates in penicillinbiosynthesis.

This paper describes the preparation and nutrientrequirements of the washed mycelium of P.chry8ogenum, and shows that the precursors ofpenicillin in complete fermentations are used in asimilar way by washed mycelium.

EXPERIMENTALMany ofthe experimental methods used have been describedin previous communications from this Laboratory (Arnstein& Grant, 1954a, b).

* Part 3: Arnstein, Clubb & Grant (1954).t Present address: Bacteriology Department, The

University of Queensland, Brisbane, Australia.