Stimulation of propionate metabolism by monocarboxylic acids

Stimulation of propionate metabolism by monocarboxylic acids

VOLATILE FATTY ACID METABOLISM 137 REFERENCES 1 S, R. ELSDEN AND A. T. PHILLIPSON, Ann. Rev. Biochem., 17 (1948) 705. 2 R. CLARK AND J. R. MALAN, On...

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VOLATILE FATTY ACID METABOLISM

137

REFERENCES 1 S, R. ELSDEN AND A. T. PHILLIPSON, Ann. Rev. Biochem., 17 (1948) 705. 2 R. CLARK AND J. R. MALAN, Onderstepoort J. Vet. Research, 27 (1956) i o i . 3 I. G. JARRETT AND B. J. PORTER, Nature, 166 (i95o) 515 . 4 R. B. JoHNsoN, Cornell Vet., 45 (1955) 273. R. J. PENNINGTON, Bioehem. dr., 5I (1952) 251. 6 R. I. PENNINGTON AND \¥. I-I. PFANDER, Biochem. J., 65 (1957) lO9. 7 K. SETO, T. TSUDA AND M. UMEZU, Tohohu J..dgr. Research, 6 (1955) 91. s D. G. ARMSTRONG, K. L. BLAXTER AND N. McC. GRAHAM,Brit. J. Nutrition, I I (I957) 392. 9 W. C, STADIE AND B. C. RIGGS, dr. Bwl. Chem., 154 (1948) 687. 10 VV~. V~'. UMBREIT, R. ]7I. BORRIS AND J. F. STAUFFER, Manometric Techniques, 3rd ed., Burgess, Minneapolis, Minn., 1957, p. 149. 11 H. BUSCH, R. B. ]71URLBERT AND V. R. POTTER, J. Biol. Chem., 197 (1952) 717 . 12 p. B. HAWK, B. L. OSER AND ~V. I-I. SUMMERSON, Practical Physiological Chemistry, I 3 t h ed., Tile Blakeston Company, New York, New York, 1954, p. 63. 13 F. \~:. DENISON AND E. F. PHARES, Ann. Chem., 24 (1952) 1628. 14 j. KATZ AND I. L. CHAIKOFF, J. Biol. Chem., 206 (1954) 88715 L. A. GREENBERG AND D. LESTER, J. Biol. Chem,, 154 (1944) 177. 18 R. J. PENNINGTON, Biochem. dr., 65 (1957) 534. 17 R. D, MCCARTHY, J. C. SHAW AND S. LAKSHMANAN, Proc. Soc. Exptl. Biol. 2~/Ied., 99 (1958) 56o. is A. L. GRAFFLIN AND D. E. GREEN, J. Biol. Chem., 176 (I948) 95. 19 E. J. MASORO, J. M. FELTS, S. S. PANAGOS AND D. RAPPORT, Arch. Biochem. Biophys., 68 (I957) 270. 2o R. J. PENNINGTON AND J. M. ArPLETON, Biochem. J., 69 (I958) I19. 21 p. HELL, J. Biol. Chem., 206 (1956) 671. 22 H. ]~EINERT, D. E. GREEN, P. HELL, ]71. ]7lIFT, R. V¢'. VAN KORFF AND C. V. RAMAKRISHMAN, dr. Biol. Chem., 203 (1954) 35. 23 M. E. JONES AND F. LIPMANN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 590. 24 F. FRIEDBERG, J. ADLER AND ]7I, A. LARDY, dr. Biol. Chem., 219 (I956) 943.

Biochim. Biophys. Acta, 41 (I96O) I3O-I37

STIMULATION

OF P R O P I O N A T E

BY MONOCARBOXYLIC

METABOLISM ACIDS*

G. I. P R I T C H A R D * * AND S. B. T O V E

Animal Nutrition Section, Department o/Animal Industry, North Carolina State College, Raleigh, N.C. (U.S.A.) (Received S e p t e m b e r 28th, I959)

SUMMARY

Butyrate stimulates the metabolism of propionate in liver from rats and non-fasted sheep, though to a lesser extent than fasted sheep. The butyrate stimulatory effect is exerted on the carboxylation of propionate but not propionyl-CoA. Isobutyrate, fl-hydroxybutyrate, valerate, hexanoate and octanoate all stimulate propionate metabolism, but citrate, succinate and a-ketoglutarate are ineffective. It is postulated that the stimulative effect operates via a CoA transphorase. * Published w i t h the a p p r o v a l of the Director of Research, N o r t h Carolina Agricultural E x p e r i m e n t Station, as P a p e r No. lO74 of the J o u r n a l Series. ** P r e s e n t address: Division of Animal H u s b a n d r y , D e p a r t m e n t of Agriculture, Ottawa, Ontario, Canada.

Biochim. Biophys. Acta, 41 (196o) 137-145

I38

G. 1. PRITCHARD, S. B. TOVE INTRODUCTION

In the preceding paper 1 it was shown that butyrate exerts a marked stimulative effect on the metabolism of propionate by liver slices from fasted sheep. A stimulatory effect is surprising since the usual effect of one acid on another is that of inhibition 2-5. The results of further investigations into the nature of the stimulative effect are presented in this paper.

MATERIALS AND METHODS

Tissue preparations and incubation procedure Liver from adult sheep and adult rats was used in this study. Tissue slices were prepared and handled as previously described 1. Acetone powders of sheep liver were prepared according to the method of MORTONG. Incubations of liver slices were carried out in 25o-ml Warburg flasks as previously described 1. The amounts of the various substrates used were equalized on the basis of acetate units contributed to the citric acid cycle. The labeled substrates used were [i-~4C]propionate and ~14C]bicarbonate. Propionyl-CoA and butyryl-CoA were prepared from their respective anhydrides and coenzyme A according to the method of SIMON AND SHEMIN7. These preparations were stored at - - 1 5 ° until used.

Isolation and counting procedures In the experiments using labeled bicarbonate, the residual bicarbonate was removed at the end of the incubation b y acidifying with trichloroacetic acid and then flushing with carbon dioxide for 5 rain. Aliquots of the trichloroacetic acid supernatant were plated directly onto aluminum planchets, dried under an infrared lamp and the amount of 1'C fixed during the incubation measured. In some experiments the dicarboxylic acid fraction of the incubation medium was isolated as described 1. Fixation of inorganic phosphate was determined b y measuring the incorporation of ~2p from labeled ortho-phosphate into the nucleotides. The nucleotides were isolated by absorption onto charcoal and eluted with 5 % aqueous pyridine s. The pyridine eluents were plated onto pyrex planchets and dried under an infrared lamp. The relative counts from s"P were determined after placing an e m p t y aluminum planchet over the pyrex planchet containing the sample. This procedure blocked all of the counts from 14C and measured only the counts from 32p. Hydroxamic acid derivatives of the acyl-ester compounds in the incubation medium were prepared as described by ARONOFF9. These derivatives were separated by paper chromatography using octyl alcohol-formic acid-water (3 : I : 3) as the solvent system 1°. Control and carrier derivatives were prepared from propionic anhydride and butyric anhydride. The spots on the paper were developed b y spraying with ferric perchlorate solution. The area of the propionyl-hydroxamates was cut from the paper and counted.

Biochim. Biophys. Acta, 41 (196o) 137-145.

STIMULATION OF PROPIONATE METABOLISM

139

RESULTS

Egect of butyrate on propionate metabolism by liver slices from non-fasted sheep In the previous studies 1 it was found that butyrate exerted a marked stimulation on the metabolism of propionate from liver slices obtained from sheep starved 6-8 days. Preliminary work had revealed that this period of deprivation resulted in a drop in the liver carbohydrate from about 5 % to 1%. Since the addition of glucose to the starved liver slices also increased the metabolism of propionate, it was of interest to ascertain whether the butyrate stimulatory effect would be found in tissues of non-fasted animals. The results of adding butyrate as a cosubstrate to propionate with liver slices from a non-fasted sheep are shown in Table I. It is clear from these results that liver from fasted sheep is not required to show the stimuiatory effect of butyrate on propionate metabolism. However, the magnitude of the stimulation that was obtained in this experiment was not as great as that obtained when tissue from fasted animals was used 1. TABLE I E F F E C T OF B U T Y R A T E ON T H E METABOLISM OF [ I - 1 4 C J P R O P I O N A T E BY LIVI~R SLICES OF NON-FASTED SHEEP

All flasks contained 2oo/zmoles of propionate containing i /~C carboxyl-labeled propionate. The control flasks contained an additional 200 itmoles of propionate and the cosubstrate flasks contained ioo #moles butyrate. Incubation time, 3 h in Krebs-Ringer bicarbonate, pH 7 at 37 % Cosubstrate Fraction

None counts]rain]flask

Butyrate counts/rain/flask

CO 2

67,829 68,28o

9o,26o lOO,913

Dicarboxylic acids

9,89o lO,61o

27,44 ° 21,3oo

Effects o/butyrate on propionate metabolism by rat-liver slices Since ruminant animals are unique in that they absorb large amounts of propionate, one might expect a species difference in the butyrate stimulatory effect. To test this hypothesis four experiments were conducted with rat-liver slices. In the first three of these experiments the rats were starved 24 h, and in the fourth the rat was fed ad libitum. The results of these experiments are shown in Table II. Although some stimulation of the formation of CO~ from propionate was obtained in the first two experiments, none was obtained in the other two. Moreover, even in tile first two experiments, butyrate did not increase the 14C incorporation in the dicarboxylic acid fraction. Therefore, although a butyrate stimulation might occur in rat tissues, the stimulation was not as consistent nor as great as that obtained with sheep-liver slices.

Effects of butyrate on carbon dioxide fixation by propionate In order to determine if butyrate stimulated propionate metabolism over the pathway involving the carboxylation of propionate n or via some other pathway, Biochim. Biophys. Acta, 41 (196o) 137-14.~

140

C;. I. PRITCHARD, S. B. TOVE TABLE II EFFECT

OF B U T Y R A T E

ON T H E

M E T A B O L I S M OF

[I-14CTPROPIONATE

BY RAT-LIVER SLICES

All flasks contained 3 g liver slices in K r e b s - R i n g e r bicarbonate buffer p H 7.0 and 200 #moles p r o p i o n a t e containing I l,C ~1-14C]propionate. The control flasks contained an additional 200 ttmoles p r o p i o n a t e and i o o / z m o l e s b u t y r a t e was added to the c o s u b s t r a t e flasks. All flasks were incubated at 37 ° for 3 h, except those from E x p t . 3 which were incubated 1. 5 h, The r a t for E x p t . 4 was not fasted. Cosubstrate Fraction

Expt. No.

None counts/rain/flask

Butyrah, counts/rain/flask

CO 2

I 2 3 4

15,291 213,333 ~4,665 2o,616

33,675 376,470 14,o12 24,348

Dicarboxylic acid

1 2

26,980 12,49o

26,850 16,44o

an experiment was conducted using both I*C labeled bicarbonate and II-1*Clpropionate. The presence of butyrate stimulated the fixation of 1'C from labeled bicarbonate in addition to the incorporation of laC from [I-14C]propionate into the dicarboxylic acid fraction (Table III). These findings coupled with the fact that the butyrate stimulatory effect is found irrespective of the metabolic fraction measured 1, strongly suggests that the site of stimulation must occur early in the propionate metabolic pathway. These findings also substantiate those of BLACK AND KLEIBER12 and PENNINGTON AND SUTHERLAND la w h o suggested that, in the ruminant, propionate was carboxylated to succinate. TABLE III EFFECT IN

THE

OF B U T Y R A T E

DICARBOXYLIC

ON T H E

ACID

FIXATION

FRACTION

OF

BY LIVER

LABELED

CARBON DIOXIDE

SLICES FROM

FASTED

SHEEP

E a c h flask contained 3 g liver slices in K r e b s - R i n g e r bicarbonate p H 7.0 and 2o0 #moles propionate. I n c u b a t i o n t i m e was 3 h at 37 C Control flasks contained an additional 200/~inoles propionate and the c o s u b s t r a t e flask, i o o / , m o l e s b u t y r a t e . The labeled bicarbonate flasks contained 5 1'C ~14CINaHCOa and the labeled p r o p i o n a t e flasks, i /~C q-14Clpropionate. Labeled substrale Cosubstrah,

None Butyrate

HCOa

Propionate

counts~rain/flash

counts~rain/flask

7,32o ~9,36o

4,o52 16,646

Contribution of energy from cosubstrates in the presence of propionate ~'RIEDBERG ~1 al. 14 obtained a stimulation by hydroxybutyrate on tile carboxylation of propionate, but showed that the cosubstrate merely supplied the energy necessary for carboxylation of the propionate. I t was doubted that a similar phenomenon could account for the butyrate stimulatory effect with sheep liver since ]?iochim. Biophys. Acht, 41 (196o) 137 i45

STIMULATION OF PROPIONATE METABOLISM

141

citrate gave no stimulatory effect, yet was oxidized as rapidly as butyratO. In order to further eliminate the energy hypothesis, two experiments were conducted in which the energy production by various substrates in the presence of propionate was determined. The energy production was estimated by measuring the amount of E~2Plortho-phosphate fixed in the nucleotide fraction. In addition,/3-hydroxybutyrate, succinate and a-ketoglutarate were tested for their capacity to stimulate propionate metabolism. The results of these experiments are shown in Table IV. Of the substrates tested, stimulation of propionate metabolism was obtained only with butyrate and fl-hydroxybutyrate. No stimulation was obtained with a-ketoglutarate and an inhibition was obtained with citrate and succinate. In the previous studies 1 with liver from fasted animals, citrate neither stimulated nor inhibited propionate metabolism. It is conceivable that the explanation for the inhibition obtained in these experiments rests with use of tissue from non-fasted animals. It is interesting that adding citrate as a second cosubstrate abolished the stimulatory action of butyrate, and the corollary that butyrate did not overcome the inhibition of citrate. The results of the determination of a2p in the nucleotide fraction show that the compounds which failed to stimulate propionate metabolism, citrate, succinate and a-ketoglutarate, promoted, if anything, an increase in the amount of inorganic phosphate esterified. These findings fully substantiate the hypothesis that the mechanism of the butyrate stimulation is not simply an energy effect. T A B L E IV EFFECT OF COSUBSTRATES ON THE METABOLISM OF PROPIONATE AND THE FIXATION OF INORGANIC PHOSPHATE E a c h flask c o n t a i n e d 3 g liver slices f r o m non-fasted sheep in K r e b s - R i n g e r b i c a r b o n a t e and 2 0 0 / z n l o l e s propionate, i #C ~i 14C~propionate and i # m o l e K H z P O 4 c o n t a i n i n g 7 b*C 32p. The c o n t r o l flask c o n t a i n e d an a d d i t i o n a l 200 # m o l e s propionate. The cosubstrates a m o u n t s were b u t y r a t e and/5 h y d r o x y b u t y r a t e , i o o / , m o l e s ; citrate, succinate and a - k e t o g l u t a r a t e , 2 o o / z m o l e s . I n c u b a t i o n w a s at 37 ° for 1.5 h. Cosubstrate

Expt.

taCO ~ counts]min/flask

32p fixed counts/rain/flask

None

5 6

33, o18 23,676

396 382

Butyrate

5 6

44,264 50,602

394 355

fl-hydroxybutyrate

5 6

64,814 78,765

517 246

Citrate

5 6

13,°1° 8,455

660 457

Succinate a-ketoglutarate

5 6

7,452 21,789

475 617

Butyrate +citrate

5 6

lO,932 5,587

562 369

Effect of isobutyrate, valerate, hexanoate and octanoate on propionate metabolism Heretofore stimulation of propionate metabolism has been observed with Biochim. Biophys. Acta, 41 (196o) 137-145

142

G . I . PRITCHARD, S. B. TOVE TABLE V STIMULATION OF PROPIONATE METABOLISM BY MONOCARBOXYLIC ACIDS

All flasks contained 3 g liver slices in 20 ml K r e b s - R i n g e r bicarbonate p H 7.0 and 200/*moles p r o p i o n a t e containing i /*C [i 14C]propionate. All c o s u b s t r a t e s were added at a level of lOO/*moles. An additional 200/*moles p r o p i o n a t e was added to the control flask. I n c u b a t i o n was for 3 h at 37 °. I n c u b a t i o n was not initiated until several h o u r s after the a n i m a l was sacrificed. Cosubstrate

~C in C02 counts/rain/flask

None Butyrate Isobutyrate Valerate Hexanoate Octanoate

3,92 i I o, 486 6,634 I o, 663 5, t 48 5,545

monocarboxylic acids, acetic and butyric, but not with polycarboxylic acids, citric, succinic and a-ketoglutaric. In order to ascertain if the stimulation would be found with other monocarboxylic acids, an experiment was conducted with liver slices from fasted sheep using as cosubstrates isobutyrate, valerate, hexanoate and octanoate. All of the cosubstrates tested stimulated the incorporation of 14C from carboxyllabeled propionate into carbon dioxide, but two degrees of stimulation are apparent (Table V). Isobutyrate, hexanoate and octanoate all stimulate to the same degree, but this is less than the stimulation obtained with valerate and butyrate. It is significant that both valerate and isobutyrate exert a stimulatory effect on propionate metabolism even though they are propionate precursors 15,16.

Effect of butyrate on the metabolism of propionyl-CoA and on the metabolism of propionate plus CoA The results of previous experiments 1 have suggested that the stimulatory effect of butyrate seems to be exerted at an early stage in the metabolism of propionate. In order to determine if butyrate stimulated the metabolism of propionate that had been TABLE VI EFFECT OF BUTYRATE ON THE FIXATION O F 14CO2 IN THE PRESENCE OF PROPIONATE AND PROPIONYL-CoA The e n z y m e s y s t e m was extracted from sheep-liver acetone p o w d e r w i t h io volumes of o.1 M tris buffer, p H 8.o. The i n c u b a t i o n m i x t u r e consisted of tris buffer, p H 8.o, 5 o / , m o l e s ; MgC1z, 5 / , m o l e s ; GSH, 4 / , m o l e s ; ATP, 4/*moles; K H C O a, 5 #moles,; Nal4CO3, 3/*moles, o.9/*C; and t h e enzyme. All s u b s t r a t e s were added in o. 5/*mole quantities. I n c u b a t i o n s were carried o u t in 4-ml test t u b e s at 3 °o for 2o rain u n d e r air. The reactions were stopped b y adding o. 5 ml of io O//o trichloroacetic acid. Values given are the m e a n of duplicate t u b e s except the t u b e with propionate and butyryl-CoA. Additions

P r o p i o n a t e + CoA P r o p i o n a t e + CoA + b u t y r a t e P r o p i o n a t e + butyryl-CoA Propionyl-CoA Propionyl-CoA + b u t y r a t e Butyryl-CoA

~4Cfixed counts/rain/tube

3,753 5,798 6,749 8,704 9,788 402

Biochim. Biophys. Acta, 41 (196o) 137-145

STIMULATION OF PROPIONATE METABOLISM

143

activated with coenzyme A, an experiment was conducted using a crude extract of sheep-liver acetone powder, the results of which are shown in Table VI. The fixation of 14C by propionate from 1~C02 was used as the criterion. The presence of butyrate and butyryl-CoA increased the carboxylation of propionate 54 % and 80 %, respectively. Although replacing the propionate and coenzyme A with propionyl-CoA resulted in more than doubling the CO~ fixation, the addition of butyrate to propionyl-CoA increased the CO 2 fixation only 12 %. The small amount of ~4C fixed by the system in the presence of butyryl-CoA, but the absence of propionate provides evidence that the COz fixation in the system employed was primarily the result of carboxylation of propionate. These results are suggestive TABLE VII EFFECT OF BUTYRATE AND BUTYRYL-CoA ON THE FORMATION OF PROPIONYL-CoA FROM [I-14C~PROPIONATE T h e e n z y m e s y s t e m was an e x t r a c t of sheep-liver acetone p o w d e r b y IO v o l u m e s of o.i M Tris buffer, pt-I 7.5. I n c u b a t i o n m i x t u r e contained 5 #raoles MgCI~, 4 t traoles GSH, 4 / t m o l e s ATP, 20 ttmoles p r o p i o n a t e containing 0. 5 #C 14C and the enzyme. Additions of 0. 5/zmole CoA, b u t y r a t e or b u t y r y l - C o A were m a d e where indicated. I n c u b a t i o n s were for IO rain a t 3 o°. Reactions were s t o p p e d b y adding 0.2 inl of o.i M h y d r o x y l a m i n e in ethanol, Additions

[l*C] Propion~I-CoA counts]rain

CoA B u t y r a t e + CoA Butyryl-CoA

i43 14o 167

that a transfer of coenzyme A may be involved in the stimulation of propionate metabolism by butyrate.

Transfer of coenzyme A. In this experiment an attempt was made to obtain information on the transfer of CoA from butyryl-CoA to propionate. A Tris buffer extract of sheep-liver acetone powder and IIA4C]propionate were incubated with and without butyryl-CoA. The hydroxamate derivative of propionyl-CoA was separated by chromatography and counted. The results are given in TableVII. Unfortunately, the results are inconclusive. The presence of butyryl-CoA resulted in a slight increase in the 14C found in propionylCoA, but the presence of butyrate was without effect. The radioactivity recovered in the butyryl-CoA was negligible. DISCUSSION

When one fatty acid is added to a system metabolizing another fatty acid, the usual effect observed is that of inhibition ~, a. Thus the stimulation of propionate metabolism by monocarboxylic acids is somewhat unique. Although this stimulatory effect is not necessarily confined to starved ruminant liver, the magnitude of the stimulation is greater when starved sheep liver is used than when either rat liver or non-starved sheep liver is employed. Species difference with respect to the capacity for metabolism of volatile fatty acids has been reported by PENNINGTON AND APPLETONs. These Biochim. Bi ophy s . Acta, 41 (196o) I 3 7 - I 4 5

144

c~. I. PRITCHARD, S. B. TOVE

investigators also reported 5 that the conversion of propionate to CO2 was not altered by acetate. It is possible that the stimulatory effect was not observed because the animals used were not starved. Since the stimulatory effect is found with butyrate, isobutyrate, valerate, hexanoate, octanoate (Table V),/3-hydroxybutyrate (Table IV) and acetate 1, but not with citrate, a-ketoglutarate or succinate (Table IV); it is apparent that the stimulatory effect on propionate metabolism is confined to the monocarboxylic acids. The finding that butyrate stimulates tile fixation of 1'C from 14CO2 as well as from ~I-14C~propionate suggests that butyrate stimulates propionate metabolism over the pathway of carboxylation to succinate. Prospects of a condensation reaction between propionate and butyrate or between propionate and acetate are unlikely because propionate failed to stimulate the metabolisme of these compounds 1. The evidence presented here indicates that metabolism of butyrate contributing the energy necessary for the carboxylation of propionate 14, thus stimulating its metabolism, is not adequate to explain the interrelationship between the two fatty acids. The butyrate stimulatory effect upon propionate was observed in liver slices in which a shortage of ATP would appear improbable. Furthermore, acids that failed to stimulate the metabolism of propionate were metabolized as rapidly as butyrate and fixed inorganic phosphate equally as well. In view of the fact that butyrate stimulates the carboxylation of propionate and that the acids of the citric acid cycle are highly labeled 1, it is probable that butyrate exerts its influence early in the metabolism of propionate. The difference in the degree of butyrate stimulation on propionate plus CoA and propionyl-CoA (Table VI) is further proof of this hypothesis and indicates that the site of stimulation is prior to the formation of propionyl-CoA. An hypothesis advanced to explain the butyrate stimulato~y effect upon propionate metabolism can be based on tile three following reactions: Propionate 4~ ATP + CoA ~ Propionyl-CoA + AMP + PP Butyrate + ATP + CoA --+ Butyryl-CoA + AMP + PP Butyryl-CoA + propionate ~ Propionyl-CoA + butyrate

(1) (2) (3)

The first is the activation of propionate to form propionyl-CoA n. The second involves the activation of butyrate to form butyryl-CoA17,18. The third, a reaction not yet reported for animal tissues, involves the transfer of the coenzyme A moiety from butyryl-CoA to propionate. This last proposed reaction is similar to the transphorase reaction 19 in which coenzyme A is transferred from one f a t t y acid to another in Clostridium kluyveri. Since the enzyme systems that activate propionate show at best only marginal activity toward butyrate 2°, 21, and the butyrate activating enzyme is inactive toward propionate ~7, it is possible that the activation of propionate could occur less readily than the activation of butyrate. Such effects could arise from differences in the rates of metabolism of the two enzyme systems and/or from the presence of a propionate specific deacylase. If one postulates that the activation of butyrate is more rapid than the activation of propionate, it is conceivable that a net reaction involving the activation of butyrate with coenzyme A and tile transfer of coenzyme A from butyryl-CoA to propionate could occur more rapidly than the direct activation of propionate. There would be essentially no difference between the direct and indirect systems with respect to energy required for propionate activation. Biochim. Biopt~ys. Acta, 41 (196o) I37 145

STIMULATION OF PROPIONATE METABOLISM

145

Such an hypothesis is in accord with the findings in the studies of the stimulatory, effect of butyrate on propionate metabolism. It would be expected, as was observed, that butyrate would stimulate the fixation of C02 and propionate plus CoA but have no effect on propionyl-CoA. No stimulation was obtained with acids of the citric acid cycle (Table IV) as would be expected. In contrast, stimulation was found with the addition of fl-hydroxybutyrate, isobutyrate, valerate, hexanoate and octanoate (Table V) and all of these acids are activated by the butyrate activating systemlL Similarly, if the proposed transphorase does exist, it must be rather non-specific for the monocarboxylic acids from 3-8 carbon atoms. Of course, this hypothesis would rest on much firmer ground if the existence of a CoA-transphorase could be established by isolation. The experiment on the isolation of E14Clpropionyl-CoA from the reaction of EI-14C]propionate with butyryl-CoA although inconclusi,, e is at least consistent with the existence of a CoA transphorase. Further work along these lines is in progress. REFERENCES I. PRITCHARD AND S. B. TOVE, Biochim. Biophys. Acta, 41 (196o) 13o. AVIGAN, J. H. QUASTEL AND P. G. SCHOLFIELD, Biochem. J., 60 (1955) 329. J. PENNINGTON AND W. H. PFANDER, Biochem. J., 65 (1957) lO9. J. PENNINGTON, Bioehem. J., 65 (1957) 534. J. PENNINGTON AND J. M. APPLETON, Biochem. J., 69 (1958) 119. K. MORTON, in S. P. COLOWlCK AND N. O. [{APLAN, Methods in Enzymology, Vol. I, Academic Press, Inc., N e w York, 1955, p. 25. 7 E. J. SIMON AND D. SHEMIN, J. Am. Chem. Soc., 75 (1953) 2520. 8 R. t{. CRANE AND F. LIPMANN, J. Biol. Chem., 2Ol (1953) 235. 9 S. ARONOFF, Techniques o] Radiobiochemistry, Iowa State College Press, Ames, 1956, p. 12o. 16 A. R. THOMPSON, Australian J. Sci. Research Ser. B, 4 (1951) 18o. 11 M. FLAVIN AND S. OCHOA, J. Biol. Chem., 229 (1957) 965. in A. L. BLACK AND M. KLEIBER, J. Biol. Chem., 232 (1958) 203. 13 R. J. PENNINGTON AND T. M. SOTHERLAND, Biochem. J., 63 (1956) 618. 14 F. FRIEDBERG, J. ADLER AND H. A. LARDY, J. Biol. Chem., 219 (1956) 943. is \V. G. ROBINSON, R. NAGLE, ]~. I~. BACHHOVVAT,F. P. I~UPIECKI AND M. J. COON, J. Biol. Chem., 224 (1957) i. 16 A. L. GRAFFLIN AND D. E. GREEN, J. Biol. Chem., 176 (1948) 95. 17 H. R. MAHLER, S. J. ~vVAKILAND R. M. BOCK, J. Biol. Chem., 204 (1953) 453. IS C. H. L. PENG, Biochim. Biophys. Acta, 22 (1956) 42. 19 E. R. STADTMAN, J. Biol. Chem., 203 (1953) 5Ol. 20 H. ]~EINERT, D. E. GREEN, P. HELE, H. HEFT, R. W. VANI~ORFF AND C. V. RAMAKRISHMAN, J. Biol. Chem., 203 (1954) 35. ~1 M. E. JONES AND F. LIPMANN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, Inc., New York, 1955, p. 590. 1 G. 2 j. 3 R. 4 R. 5 R. 6 R.

Biochim. Biophys. Acta, 41 (196o) 137-145