ROLEOF SUCCINATE AS PRECURSOR … IN PROPIONIC ACID FERMENTATION Propionate +ATP+CoA 11 CH;-CH2-C-CoA COOH-CH2-CH2COOH CO2 ±+C,-C, 0 HOOC-CH2-CH2-C-CoA CH3-CH2-COOH Suiccinate +ATP+CoA

THE ROLE OF SUCCINATE AS A PRECURSOR OF PROPIONATE INTHE PROPIONIC ACID FERMENTATION'

HARLAND G. WOOD, RUNE STJERNHOLM, AND F. W. LEAVERDepartment of Biochemistry, School of Medicine, Western Reserve University, Cleveland, Ohio, and

Department of Biochemistry, School of Veterinary Medicine, University of Pennsylvania,Philadelphia, Pennsylvania

Received for publication December 22, 1955

The mechanism of the formation of propionateby the propionic acid bacteria has remained un-certain for many years but current evidence indi-cates its formation is by decarboxylation ofsuccinate (review by van Niel, 1952). Johns(1951a) demonstrated that Micrococcus lactily-ticus decarboxylates succinate very rapidly topropionate. Delwiche (1948) and Johns (1951b)have concluded that propionate is formed by asimilar reaction with organisms belonging to thegenus Propionibacterium; however, in this casethe decarboxylation of succinate is much slowerthan with M. lactilyticus.Barban and Ajl (1951) studied the conversion

of C"'02 and propionate-C4 to C4 dicarboxylicacids by propionic acid bacteria and found incarrier type experiments that the succinate ac-quired radioactivity much more rapidly than didthe fumarate or malate. They concluded thatCO2 and propionate are converted reversibly tosuccinate without prior conversion to fumarateand malate.

Recently detailed studies of the mechanism ofthe decarboxylation of succinate to propionateand CO2 have been carried out by Whiteley(1953a, b, c) with extracts of M. lactilyticus. Shehas found that Mg++, adenosine triphosphate,and Coenzyme A stimulate the reaction. Similarresults have been obtained by Delwiche et al.(1953, 1954). The mechanism shown in figure 1illustrates some of the current views on the reac-tion. Delwiche et al. (1953, 1954), found with anenzyme preparation of Propionibacterium pento-saceum that propionate-2-C"4 is fixed in succinate

1 This work was supported by grants from theAtomic Energy Commission under Contract Num-ber AT(30-1)-1050, from the Department ofHealth, Welfare and Education, Grant Number3818, and by the Elizabeth Prentiss Fund, WesternReserve University. The C14 was obtained onallocation from the Atomic Energy Commission.

much more rapidly than is C'402 and Phares andCarson (1955) believe that there is a separateenzyme for conversion of the CO2 to Cl.Leaver et al. (1955) observed with Clostridium

propionicum that lactate-3-C14 is converted topropionate without randomization of the C14 inthe propionate. Obviously with C. propionicumfree succinate is not the precursor of the propio-nate, since a symmetrical C4 compound wouldlead to appearance of C14 in both the a and ,positions of propionate. On the other hand withpropionibacteria in similar experiments the C14was randomized in the propionate formed fromlactate-3-C 4. In spite of these differences it ispossible that propionate is formed by the pro-pionic acid bacteria in the same manner as by C.propionicum. In the case of propionic acid bac-teria it is possible that the C14 is secondarilyrandomized by reversible conversion to succinateor by some other mechanism. We have investi-gated this possibility using resting cells of Pro-pionibacterium arabinosum with propionate-3-C14 and propionate-1,3-C'4. Evidence has beenobtained that suggests that propionate and suc-cinate may be formed by more than one pathway.

METHODS

Labeled propionate was synthesized as de-scribed by Leaver et al. (1955). The succinate-2-or 3-C14 was a gift of H. E. Swim, Western Re-serve University School of Medicine.

In all the fermentations, twice-washed suspen-sions of P. arabinosum were incubated anaerobi-cally at 30 C and except where noted the cellswere grown in a glycerol-yeast extract mediumas described by Wood et al. (1955). The methodsof separation and degradation of the acids like-wise were similar to those employed formerly.

In experiments in which "intracellular" and"extracellular" compounds were separated (tables3 and 4) 100 to 150 g of cells were harvested witha Sharples centrifuge. The cells were washed

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Propionate + ATP + CoA

11CH;-CH2-C-CoA COOH-CH2-CH2COOH

CO2 +C, -C,

0

HOOC-CH2-CH2-C-CoA CH3-CH2-COOH

Suiccinate + ATP + CoA

Figure 1. The interconversion of propionate and succinate

twice by centrifugation, each time using 3 Lof 0.05 M phosphate buffer pH 6.0 and were thensuspended in 400 ml of the phosphate buffer.This mixture wasshakenina 2-L Erlenmeyerflaskin a bath at 30 C under an atmosphere of helium.After temperature equilibrium and anaerobicconditions had been established, the propionate-C'4, lactate, succinate, and L-malate were addedin 100 ml of buffer consisting of 50 ml of the 0.05M phosphate buffer and 50 ml 0.1 M NaHC03.The mixture was incubated for 5 min and then40 ml of 1 M H3P04 was usually added to bringthe pH to 3. The solution was immediatelycooled to 0 C in a dry ice bath and the cell sus-pension was centrifuged for 20 min at 0 C. Thesupernatant solution was decanted and the acidswere separated from it by ether extraction. Theseacids were designated extracellular.The cells were washed twice with 5 volumes of

distilled water at 0 C-each centrifugation being20 min. Intracellular compounds were obtainedby boiling the washed cells in an equal volume of0.5 N H2S04 for 1 hr. In order to achieve a morecomplete recovery, the cell debris was centri-fuged and treated twice more with 0.5 N H2SO4.The acids from the combined sulfuric acid ex-tracts likewise were obtained by ether extractionand were designated intracellular. The individualcompounds were isolated as already noted exceptthat each acid was rechromatographed. In thecase of succinate the rechromatographing fol-lowed oxidation with KMnO4 which destroysany lactic acid present.

Malic acid was degraded by oxidation to acet-aldehyde and CO2. (Friedemann et al., 1927).The acetaldehyde was oxidized with dichromateand the resulting acetic acid was purified bychromatography on a celite column and then

degraded by the method of Phares (1951). Thea-carboxyl of malate was obtained by treatmentwith H2S04 (Utter, 1951).For total oxidation, the chromic acid method

of van Slyke and Folch (1940) was used. The C14was counted as CO2 in the Ballentine-Bernstein(1950) proportional counter.

RESULTS

Metabolism of propionate-C"4 and succinate-CHin the presence of a fermentable substrate. Thestability of propionate as an end product of thefermentation was investigated by adding propi-onate-C"4 to fermentations of lactate and glycerolunder conditions similar to those employed byLeaver et al. (1955).The results in table 1 show that propionate

is not a stable end product of the fermentation.In fermentation 1, in which glycerol was fer-mented in the presence of propionate-3-C , theC14 became almost completely randomized in the2 and 3 carbons of propionate. Furthermore, theC14 was incorporated into succinate and the finalsuccinate had a higher specific activity than didthe propionate. In fermentation 4, it is seen thatthe succinate-2,3-C'4 did not come to isotopicequivalence with the propionate, its specificactivity being more than 6 times that of the finalpropionate. A possible explanation for this ob-servation is that labeled succinate does not enterthe cells at a rapid rate and thus the propionateremains with relatively low activity. On the otherhand, in fermentation 1 the added propionateappears to enter the cells rapidly, where it isincorporated into succinate that is being formedfrom glycerol. At the same time the specific ac-tivity of the propionate is reduced by unlabeledpropionate being generated from unlabeled glyc-

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TABLE 1The metabolism of propionate-C14 and succinate-C14 by Propionibacterium arabinosum strain 34W during

the fermentation of lactate or glycerolNote: Values in italics are in per cent of the total activity in the compound, those in bold face are

specific activity as cpm per pmole of carbon. Values in parentheses are the sum of the activities ofcarbons from degradation.

Propionate SuccinateNo. Substrate Molarity Oxid. Oxid_

CH&- CHM- COOH OCxd3 (CHr- COOH)s Cs4

1* Glycerol 0.10 56.4 48.6 0.0 0.86 45.5 4.5 1.178.65CHs-CH,-COOH 0.037 0.486 0.375 0.0 (0.86) 0.54 0.054 (1.19)

2 Lactate 0.07 48.9 49.9 1.1 3.06 - - |4.75 4.90CH,-CH,-COOH 0.025 1.46 1.49 0.033 (2.98) - - -

3 Glycerol 0.10 99.8 0.70 1.894.75 4.90CH,-CH1-COOH 0.025 1.04 0.02 (2.10)

4 Glycerol 0.10 49.7 48.7 1.6 - - - -15.3

HOOC-CH2-CH,- 0.27 0.573 0.556 0.018 (1.147) - - 7.16COOH

5 46.1 49.8 4.1 5.20 46.9 8.8 4.525.9CH,-CHOH-COOH 0.07 2.40 2.59 0.214 (5.20) 2.10 0.150 (4.50)

* Data from fermentation 1 were reported in part in a previous publication (Wood and Leaver,1953).

All fermentations were in 300-ml round-bottom flasks with ground-glass joints and contained 0.075 Mpotassium phosphate buffer (pH 5.9), 0.125 M NaHCOs, 5 per cent cells in addition to indicated sub-strates. The flasks were evacuated and then the components of the reaction mixture were added througha dropping funnel. Cells for fermentation 2 and 5 were grown on sodium lactate, 1.0 per cent; glucose,0.1 per cent; yeast extract, 0.5 per cent; phosphate buffer, 0.01 m; for 3 days at 30 C. Time of fermenta-tion, 24 hr-except No. 1, which was 17 hr.No. 1, Glycerol fermented = 5.60 mm. Volatile acid 5.86 mEq (1.94 mm of propionate were added),

nonvolatile acid - 1.96 mEq, C02 = -0.99 mm. No. 2, Lactate fermented = 4.27 mm. Volatile acids =4.90 mEq (1.50 mm of propionate were added), nonvolatile acid = 0.60 mEq, C00 = 1.21 mm. No. 3,Glycerol fermented = 5.60 mm. Volatile acids = 5.40 mEq (1.50 mm propionate were added), non-volatile acid = 1.98 mEq, C02 = -0.99 mm. No. 4, Glycerol fermented = 5.60 mm. Volatile acids =4.38 mEq, nonvolatile acids = 5.15 mEq (1.60 mm succinate were added), C00 = -1.29 mm. No. 5,Lactate fermented = 4.11 mm. Volatile acids = 3.85 mEq, nonvolatile acids = 0.56 mEq, C02 = 0.80mM.

Glycerol + NaHC14O, (conditions same as No. 1 except no propionate was added). Glycerol fer-mented = 3.82 mmr. Volatile acids = 2.89 mEq, nonvolatile acids = 1.12 mEq, C02 = -0.74 mm. InitialC02 = 25.2 cpm per Mmole. Final C02 - 21.3 cpm per pmole. Carboxyl of isolated propionate 4.22 cpmper pAmole.

erol. In this manner propionate radioactivity experiment (no. 1), a glycerol fermentation wasfalLs below that in the succinate even though the conducted in which NaHC"O,s was added (foot-propionate serves as the source of the C14 of the note of table 1). This was done to obtain infor-succinate. mation on the relationship of C02 fixation to the

In conjunction with the propionate-3-C4 randomization of the isotopic carbon in propio-

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nate. In fermentation 1, the randomization was 87per cent ((2 X 0.375 X 100) *. (0.485 + 0.375))but in the glycerol-NaHC"4O, experiment only19.8 per cent of the carboxyl acquired activityfrom the CO2 (4.22 X 100 . 21.3 = 19.8, foot-note of table 1). The lack of equivalence betweenthe randomization reaction and the fixation ofC02 will be discussed further in relation to theexperiments of table 2. The results are verysimilar to those reported by Delwiche et al.(1953) in which it was found that propionate-2-C14 is fixed in succinate more rapidly than is C02.

In experiments no. 2 and no. 3, propionatewithequal labeling in the 2 and 3 positions was usedin order to investigate whether this compoundis converted to a symmetrical C3 compound.Leaver et al. (1955) found that lactate-3-Cl4 wasconverted to propionate containing more activityin the 2 position than in the 3 position and withconsiderable activity in the carboxyl group. Theyconsidered that such a distribution might ariseif the C14 were randomized via symmetrical C4and C, intermediates (Leaver and Wood (1953)give illustrations). Using propionate containingequal activities in the 2 and 3 carbons the ratioof the activities in the two carbons would not bechanged by reversible conversion to a symmetri-Cal C4 dicarboxylic acid but would be changedby reversible conversion to a symmetrical Cs.In none of the experiments of table 1 was theresignificant conversion of the 2 and 3 positionsof propionate to the carboxyl (of propionate)and in fermentation 2, the ratio of activities inthe 2 and 3 carbons was not changed. Thus therewas no evidence that propionate was convertedto a symmetrical C3 compound. In the controlfermentation (no. 5) there were indications ofthe occurrence of a symmetrical C0 compoundfrom lactate-3-C"4, since some radioactivityappeared in carbon 1 and there was a higherspecific activity in carbon 2 than in carbon 3.It has been our experience that large differencesbetween the 2 and 3 carbons of propionate, suchas reported in fermentation 2a (table 2) andfermentations 6 and 9 (table 3) in the paper byLeaver et al. (1955), are not obtained con-sistently.

Interaction among the fermenation end prod-uct8, propionate, succnate, acetate, and CO2 in theabsence of a fermentable substrate. Fermentationswere next studied in which the lactate and gly-cerol were omitted from the reaction mixture.

In these experiments (table 2) 0.02 x propionate,succinate, and acetate were added to washedcells and either the propionate or succinate waslabeled.The data of the propionate-1 ,3-C14 experiment

(no. 6, table 2) are of particular interest: therandomization of the 3 position of propionateinto the 2 position was complete, but there wasvery little loss of C14 from the carboxyl carbon(20.8 as compared to 19.6). The high radioactiv-ity in the carboxyl carbon as compared with thatin the a and # carbons is in itself not evidenceagainst randomization via succinate. By inspec-tion of the sc7heme in figure 1, it is evident thata small pool of C, within the cell could be inisotopic equilibrium with the carboxyls of pro-pionate and still maintain a high activity. On theother hand, the # position would be diluted 50per cent by randomization with the a position. Ifit is assumed that succinate is the only precursorof propionate, however, the ratio of the radioac-tivity in the carboxyl carbon to that in the aand # positions (average) should be the same inthe two compounds. The ratio in propionatewas 19.6/11.2 = 1.75, while in succinate it was1.18. These data make it seem very unlikely thatsuccinate was the sole source of the randomizedpropionate.The high activity remaining in the carboxyl

group of the propionate cannot be due to the factthat part of it did not react, since the randomi-zation of isotope between the 2 and 3 carbonswas complete. That the low activity in the car-boxyl of succinate was not due to loss of C14 byexchange with unlabeled C02 is evident fromexperiment 7, in which the C"O4-NaHC"O4 of themedium was labeled. Only 0.3 per cent (0.07 x100/26.0) of the carboxyls of the succinate origi-nated from C02. Furthermore, if the succinatecarboxyls had lost C14 by exchange, thepropionatewould have reflected this loss according to themechanism of figure 1.

In experiments 6, 7, and 8 the specific activitiesof the succinate were much less than those of thepropionate. This could be due to a slow intercon-version of propionate and succinate, or to slowtransport of the succinate in and out of the cells.It should be noted that the validity of the aboveconclusions is not influenced by possible permea-bility factors, provided the C14 distribution ofthe succinate is not changed during transportout of the cell.

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WOOD, STJERNHOLM, AND LEAVER

TABLE 2The interaction of propionate, succinate, acetate, and CO2 by resting cells of Propionibacterium arabinosum

Note: The values in italics are in per cent of the total activity of the compound; those in bold faceare specific activity as cpm per jmole of C. The values in parentheses are the sum of the specific ac-tivities of the individual carbons from the degradation of the compound.

Distribution of C14 in Compounds at End of Fermentation

Labeled Compound Added Propionate Succinate Acetate

CHr- CH- COOH CdOxd (CHI COOH)' Oxi4 CHs- COOH Oxid. COsCHs-CHs- CO C x 3 x2 Cx 26: 26.6 20.8 25.7 27.6 46.6 40.1 2?3.0 27.0 1.51 60.6 49.6 2.38 1.57CH-CHz2-COOH 10.8 11.6 19.6 (42.0) 0.32 0.387 (1.43) 1.01 0.99 (2.00)

7: 51.7CHx-CH2-COOH 63.5 33|. 3.3 40.0 46.1 3.8 1.75 52.2 47.8 1.16 -26.0C02 22.3 11.9 1.18 (35.9) 0.84 0.07 (1.82) 0.53 0.485 (1.01) 23.3

8:4.73 61.2 46.9 1.9 0.36 -CH3-CH2-COOH 1.17 1.07 0.04 (2.28) - - - -

9: 15.3 - - - - - 48.9 61.1 0.604HOOC-CH2-CH -- 1.93 - - 14.4 0.321 0.337 (0.658)COOH

All fermentations were in 300-ml evacuated flasks to which the components were added through adropping funnel.No. 6, 7, and 9 had the following composition: propionate, 0.02 M; acetate, 0.02 M; succinate, 0.02 M;

NaHCO:, 0.075 M; potassium phosphate buffer, 0.10 M; pH 5.9; and 4 per cent washed cells. No. 8 hadthe following composition: propionate, 0.02 M; succinate, 0.02 M; NaHCO3, 0.057 M; potassium phosphatebuffer, 0.075 M; pH, 5.9; and 5 per cent washed cells. No. 7, 8, and 9 were fermented for 40 hr., No. 6for 6 hr., No. 7 and 9 the cells were grown for 7 days at 30 C, No. 6 and 8 for 5 days at 30 C.

The experiment with labeled succinate no. 9(table 2) provides information on the possibilityof a direct cleavage of succinate to two acetatemolecules. Topper and Stetten (1954) have con-cluded from studies with deuterium and C'4-labeled succinate that succinate cleavage occursin rat liver (see also Seaman and Naschke, 1956).If succinate were converted to acetate by thismechanism the methyl position would be labeledand the carboxyl unlabeled. In the present ex-periment no. 9, it was found that the methyl andcarboxyl carbons had equal activity, whichargues against the occurrence of a central cleav-age of succinate. The acetate formed from thepropionate-3-C'4 in fermentations 6 and 7 likewisewas equally labeled.Distribution of C14 in "intracellular compounds."

It is probable, in the experiments of tables 1 and 2,that the compounds added to the medium do notcompletely equilibrate (i. e., mix uniformly) withthe same compounds present intracellularly.

Although the distribution pattern of the C14probably gives an indication of the mechanism,the isotope concentration per se is not an indica-tion of the relative quantitative significance ofthe reactions occurring. Thus the fact that thepropionate-3-C'4 was completely randomizedin experiment 6 (table 2) and the succinate ac-quired only one-thirtieth the activity of thepropionate, does not of itself prove that propio-nate is not converted to succinate rapidly. Thesuccinate produced inside the cell may acquireC14 from propionate very rapidly, but it mayequilibrate (or mix) with the extracellular suc-cinate very slowly. This weakness of the carrier-type studies has been demonstrated by Sazand Krampitz (1954) and Swim and Krampitz(1954) in their studies on the tricarboxylic acidcycle in bacteria. To avoid this difficulty, theseinvestigators isolated the naturally producedintermediates directly from large amounts ofmetabolizing cells. Their procedures have been

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used with some modification in the present stud-ies. The conditions used and the recovery ofacids are given in table 3; the C14 data are intable 4.On the basis of the recovered extracellular

acids (table 3), the propionate and acetate in-creased during the fermentation and the succi-nate, malate, and lactate decreased. In differenttrials, the yields of intracellular acids variedconsiderably. The largest yield was obtained inno. 13, in which the cells were not acidified priorto the washing. The lowest yield was obtainedin no. 11, in which case the celLs were inactivatedby rapidly dispersing them into a large volumeof water at 95 C. In no. 14, extracellular andintracellular acids were not separated.The amount of intracellular acetate was quite

large and its C14 content was low. It seems likelythat some of this acetate arose from hydrolysisof unlabeled structural components of the cells.

In fermentations 10, 11, and 12 (table 4)internal propionate had a lower C14 activitythan did the external propionate. This is re-markable, since the internal propionate pool wasthe source of the external propionate. In fermen-tation 12, where C1402 was the only labeled sub-strate, any radioactive external propionate musthave been produced metabolically (intracellu-larly).2 This also was true in fermentation 10,since the C14 of position 3 of propionate wasalmost completely randomized with position2-indicating that all the propionate had reactedand was of cellular origin. The most probableexplanation of the labeling difference betweeninternal and external propionate is that there ismore than one pool of propionate in the cell andthat one of these is of higher specific radioactivitythan the others and gives rise to extracellularpropionate more rapidly than does the low-activity pool. These pools might be free acids,coenzyme A derivatives or enzyme complexes,present in different structural components in thecell, such as mitochondria, microsomes, or the cellmembrane.Of even greater interest is the relative C14

distribution within the propionate atoms. Infermentation 10, the ratio of carboxyl to carbon2 (0.182/0.115) equals 1.58 in the internal pro-pionate and 1.25 in the external propionate. Alsothe succinates differed both in total activity and

2 The expression "intracellular" is perhaps amisnomer because the "intracellular" acids mayalso arise from the cell surface or membranes.

in the distribution of the C14. These observationssuggest that propionate and succinate probablyare formed by more than one pathway, e. g., onepathway may give rise to external acids morerapidly than do the others, thus causing theexternal acids to differ in isotope content fromthe internal acids.

In arriving at these conclusions it is assumedthat there is no change in C14 content or distri-bution in intracellular products during the timethe cells are being washed at 0 C. Fermentation 11was set up as a check on this assumption. In thiscase the reaction was stopped after the 5-minincubation by suddenly raising the temperatureto 95 C. Only very small amounts of propionlcand succinic acids (intracellular) were obtainedfrom the celLs after this treatment, and thesehad a very low radioactivity. Apparently onlythe low-activity pools remained in the cellsfollowing the treatment, and practically all theacids from the high-activity pools were lost.In this experiment it may be assumed that theendogenous metabolism was stopped promptly.It is thus reasonably certain that the C1' contentof the intracellular acids was not reduced byreactions which occurred after the high-activityextracellular acids had been removed.

In contrast to fermentation 6 (table 2), inwhich the ratio of carboxyl to 2 and 3 carbonactivity of the propionate was quite differentfrom that of the succinate, the ratios in fermen-tation 10 were similar (intracellular 1.57 and1.67, extracellular 1.23 and 1.28). In thisparticular experiment (10), the ratios consideredalone are compatible with the possibility thatsuccinate was the major source of propionate.

In the experiments of table 2 the succinateacquired activity from the propionate veryslowly. On the other hand, in the experimentswith a large mass of celLs the external succinatebecame highly labeled even though the timewas only 5 min. In the experiments of table 2the proportion of succinate to cells was 0.5 mmper g (wet weight), whereas in the experimentsof table 3 the proportion was only 0.007 mmper g of cells. It thus appears that with a largeamount of cells and a small amount of succinatethere is better equilibration between externaland internal molecular species.

In experiments 12 and 13, where the intra-cellular compounds were isolated, the resultswere quite different in the internal acids thanthose obtained by Barban and Ajl (1951), who

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WOOD, STJERNHOLM, AND LEAVER

used the carrier technique. The malate withinthe cells acquired activity very rapidly fromboth the propionate and C02, and the activitywas of the same order of magnitude as that inthe succinate. The external malate, on thecontrary, had a very low activity. It is thus quiteclear that the carrier technique and externalacids give a false picture of the role of malatein the conversions of propionate and C02.The internal malate was degraded in anattempt

to obtain some information about the mech-anisms of C02 fixation and of its formation frompropionate. If C'402 in fermentation 12 enteredmalate via fixation in the B-carboxyl of oxalace-tate or if the propionate-3-C'4 or -1,3-C'4 infermentations 10 and 13 were converted tomalate after a oxidation, C14 activity in themalate would not be symmetrical. It was foundthat the C14 activity was symmetrical withinexperimental error. This symmetry may resulteither from the reversible action of fumarasewhich would randomize the activity or becauseC02 and propionate entered malate by way ofsuccinate. Further studies will be necessarybefore any decision can be reached on this ques-tion.

DISCUSSION

There are considerable data which might beinterpreted as evidence that the decarboxylationof succinate is not the sole mechanism of pro-pionate formation. The turnover of C02 islower than would be expected if this were theonly mechanism (Wood and Leaver, 1953). Onthe other hand this could be accounted for if thedecarboxylation to propionate involves a C,other than C02. Of greater weight is the fact-that the tracer distribution patterns in the pro-pionate and succinate are not always alike aswould be expected if one were the sole precursorof the other. For example, Leaver and Wood(1953) found that succinate has a higher activityin the a , positions than does the propionatewhen formaldehyde-C14 is fixed in fermentationsof unlabeled glucose, glycerol, or pyruvate. Un-equal distributions have also been observed infermentations of different types of labeled glu-cose (Wood et al., 19-55) and with lactate-3-C0(Leaver et al., 1955).The present work provides evidence that

propionate is metabolized by a mechanism thatmay not involve succinate. With propionate-1,3-

C14 the distribution of the C14 was different in thepropionate and succinate. While this appearsincompatible with the view that the describedisotope randomization in propionate occurs bymeans of interaction with succinate, the presentwork also indicates that there is more than onepool of propionate and succinate respectivelyand that these are formed by different pathways.One pathway apparently gives rise to extracellu-lar acids more rapidly than others. It therefore ispossible for the C14 distribution of the propionateto differ from the succinate even though succinateis its sole precursor. This is true because one of thetypes of succinate-C04 may be the source of mostof the propionate.

It is of interest that Batt and Martin (1955)have found that Nocardia corallina oxidizes pro-pionate-3-C14 to pyruvate and the C'4 does notbecome equally distributed between the 2 and 3carbons of pyruvate. Thus in this case succinateis not a direct intermediate in the metabolism ofpropionate. An interesting mechanism for therandomization of the 2 and 3 carbons of pro-pionate, which would not involve losses of thecarboxyl carbon or the participation of succinateor C4-dicarboxylic acids, has been proposed byMahler and Huennekens (1953). Their mecha-nism involves a symmetrical cyclopropane deriva-tive. Recently Flavin et al. (1955) have found thatpig heart extracts supplemented with propionylcoenzyme A, adenosine triphosphate, and Mg++,fix C02 to form methylmalonate and that liverpreparations convert methyl malonate to suc-cinate. It seems possible that methyl malonatemay also be involved in some way in propionatemetabolism by propionic acid bacteria.Whether or not propionate can be formed by

direct reduction by the propionic acid bacteriathrough a mechanism similar to that occurringin C. propionicum is still not known. If directreduction did occur the observed isotope random-ization in propionate would have to be ascribedto secondary reactions. Cardon and Barker(1947) have shown that C. propionicum reducesacrylate to propionate, but a similar conversiondid not occur in tests with the propionic acidbacteria (Barker and Lipmann, 1944). However,investigations with acrylyl coenzyme A shouldbe done since this is a more likely direct reactantin the reduction. Further differences between C.propionicum and the propionic acid bacteriahave been described. Thus C. propionium does

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SUCCINATE IN PROPIONIC ACID FERMENTATION

not utilize C02 and the fermentations of lactateand pyruvate by this organism are equallysensitive to NaF (Johns, 1952). With propionicacid bacteria Barker and Lipmann (1944) foundthat NaF prevented lactate reduction underconditions in which pyruvate was reduced topropionate. Although the above evidence pointsto a different mechanism in these two organisms,the difference is not necessarily great. C. pro-pionicum may lack the ability to convert CO2 toan active form but may use the C, that is formedin decarboxylation. The exact locus of the NaFblock in the conversion of lactate to propionateby propionibacteria is unknown and thereforeits significance remains uncertain.The work of Barban and Ajl (1951), based on

isotope experiments of the carrier type, seemedto indicate that malate (and thus oxalacetate) isnot involved in C02 fixation by propionic acidbacteria. These results raised an importantquestion concerning present concepts of thefermentation. It has been assumed that pro-pionate is formed by C02 fixation with pyruvateto yield oxalacetate which is then converted tomalate, fumarate, and succinate; then the latteris decarboxylated to propionate. If the C02fixation were directly into succinate, and notinto oxalacetate or malate, the proposed pathwayof propionate formation would not be feasiblebecause the pathway to succinate via pyruvateand oxalacetate would be eliminated. Thepresent study shows that results obtained bythe carrier technique using intact cells are notreliable from the standpoint of deciding precursor-product relationship.The results emphasize the difficulty of extra-

polating from in vitro studies to in vivo conditions.Although there are cell-free enzyme systemswhich bring about the decarboxylation of suc-cinate to propionate, it appears that there alsoare other reactions by which propionate is formedin the cell. Only by a combination of all typesof in vivo and in vitro experiments can one hopeto approach an understanding of the metabolismof the whole cell.

SUMMARY

It has been demonstrated that propionate isnot an inert end product of the propionic acidfermentation. The C14 of propionate-3-C'4 israpidly randomized between the 2 and 3 carbons.This randomization is not accompanied by an

equivalent fixation of C02 and it does not appearto occur by reversible conversion to succinate.

Detection of the reversible conversion ofpropionate to a symmetrical Ca compound wasattempted with propionate-2,3-C14. No evidencewas obtained for such a conversion.

Succinate-2,3-C" was not metabolized rapidlyprobably because of slow transport into the cells.Succinate was converted to propionate and ace-tate. The acetate was equally labeled in themethyl and carboxyl carbons; thus there was noevidence of central direct cleavage of the succi-nate to two acetate molecules.

Experiments using the isotope carrier tech-nique indicated that propionate is converted tosuccinate very slowly. When intracellular acidswere examined it was found that propionate isconverted to succinate very rapidly.

Intracellular malate rapidly acquired radio-activity from labeled propionate and C02whereas the extracellular malate was labeled onlyslowly. The incorporation of C02 was about thesame in both succinate and malate in very shortincubations and thus there was no indication ofwhich, if either, of the two acids are involved inthe primary fixation. Evidence is presented forthe existence of more than one pool of propionateand succinate in the cell and it is concluded thatsuccinate and propionate are probably formedby more than one pathway.

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of propionate and succinate by Propioni-bacterium pentosaceum. J. Biol. Chem., 192,63-72.

BARKER, H. A. AND LIPMANN, F. 1944 Onlactic acid metabolism in propionic acidbacteria and the problem of oxido-reductionin the system fatty-hydroxy-keto acid.Arch. Biochem., 4, 361-370.

BATTr, R. D. AND MARTIN, J. K. 1955 Theoxidation of propionic acid by Nocardiacorallina. Proc. Univ. of Otago MedicalSchool, 33, 18-19.

BERNSTEIN, W. AND BALLENTINE, R. 1950 Gasphase counting of low energy emitters. Rev.Sci. Instr., 21, 158-162.

CARDON, B. P. AND BARKER, H. A. 1947 Aminoacid fermentation by Clostridium propioni-cum and Diplococcus glycinophilus. Arch.Biochem., 12, 165-180.

DELWICHE, E. A. 1948 Mechanism of propionicacid formation by Propionibacterium pento-saceum. J. Bacteriol., 56, 811-820.

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LEAVER, F. W. AND WOOD, H. G. 1953 Evi-dence from fermentation of labeled sub-strates which is inconsistent with presentconcepts of the propionic acid fermentation.J. Cellular Comp. Physiol., 41, Suppl. 1,225-240.

LEAVER, F. W., WOOD, H. G., AND STJERNHOLM,R. 1955 Fermentation of three carbon sub-strates by Clostridium propionicum andPropionibacterium. J. Bacteriol., 70, 521-530.

MAHLER, H. R. AND HUIENNEKENS, F. M. 1953The pathway of propionate oxidation. Bio-chim. et Biophys. Acta, 11, 575-583.

PHARES, E. F. 1951 Degradation of labeledpropionic and acetic acids. Arch. Biochem.and Biophys., 33, 173-178.

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SWIM, H. E. AND KRAMPITZ, L. 0. 1954 Aceticacid oxidation by Escherichia coli: Quantita-tive significance of the tricarboxylic acidcycle. J. Bacteriol., 67, 426-434.

TOPPER, Y. J. AND STETTEN, D., JR. 1954 For-mation of "acetyl" from succinate by rabbitliver slices. J. Biol. Chem., 209, 63-71.

UTTER, M. F. 1951 The interrelationships ofoxalacetic and L-malic acids in carbon diox-ide fixation. J. Biol. Chem., 188, 847-863.

VAN NIEL, C. B. 1952 Introductory remarkson the comparative biochemistry of micro-organisms. J. Cellular Comp. Physiol., 41,Suppl. 1, 11-38.

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WHITELEY, H. R. 1953a Cofactor requirementsfor the decarboxylation of succinate. J.Am. Chem. Soc., 75, 1518.

WHITELEY, J. R. 1953b The mechanism of pro-pionic acid formation by succinate decarbox-ylation. I. The activation of succinate.Proc. Natl. Acad. Sci. U.S., 39, 772-779.

WHITELEY, H. R. 1953c The mechanism of pro-pionic acid formation by succinate decarbox-ylation. II. The formation and decarboxyla-tion of succinyl-CoA. Proc. Natl. Acad. Sci.U.S., 39, 779-785.

WOOD, H. G. AND LEAVER, F. W. 1953 CO3 turn-over in the fermentation of 3, 4, 5 and 6carbon compounds by the propionic acidbacteria. Biochim. et Biophys. Acta, 12,207-222.

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