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Propionate oxidation by washed rabbit liver “particles”
Propionate
Oxidation
by Washed Rabbit Liver “Particles” Jack B. Wolfe’
From the Scripps Metabolic Received
Clinic, La Jolla, California
February
5, 1955
INTRODUCTION
The metabolic pathway of propionate oxidation in mammalian systems still remains to be elucidated. One possible pathway for the utilization of this compound is a direct oxidation to pyruvate. This mechanism was initially proposed for animal tissuesby Hahn and Haarmann (1) who noted an increase in pyruvate formation when propionate was incubated with muscle mince. Mahler and Huennekens (2) using a rabbit liver “cyclophorase” system also provided evidence for a modified alpha oxidation mechanism. These latter authors proposed the reaction sequence: propionate, acrylate, L-lactate, n-lactate, pyruvate, etc., to account for their experimental data. An alternate pathway in which propionate is converted into the J-carbon dicarboxylic acids by a mechanism not involving pyruvate has been suggested by isotopic experiments (3, 4). Lardy (5) considers this pathway to involve a carboxylation reaction with the formation of succinate. In the present study isotopic tracers were utilized to investigate the pathways involved in the oxidation of propionate by washed rabbit liver “particles.” METHODS
Preparation
of Washed
“Particles”
Twenty grams of freshly removed rabbit liver was minced into a Waring blendor containing 80 ml. of cold KCl-phosphate buffer solution (6 parts 0.85% KC1 to 1 part 0.1 2M Na2HP04 , pH 7.5). The mixture was blended for 30 sec. at one-half maximum speed, filtered through gauze, and centrifuged in the cold (4 f 1”) for 5 min. at 4500 X 9. The resulting particles were washed two times with 80 ml. of cold KCl-phosphate buffer solution and centrifuged at 4500 X g for 4 min. after 1 Charles
Willard
Stimsom
Postdoctoral
Fellow 414
in Biochemistry
PROPIONSTE
OXIDATION
each washing. The washed “residue” was finally suspended in a volume of cold KCI-phosphate buffer solution so that 1 ml. of the suspension represented the residue obtained from 0.66 g. of the original weight of the liver. Incubations were conducted in Warburg vessels at 37” in an air atmosphere. Oxygen ronsumption was measured according to conventional manometric procedures (6). At the end of the incubation period 50% sulfuric acid was added to stop the reaction and to release carbon dioxide from the reaction mixture. The respiratory carbon dioxide was collected in alkali and then precipitated as B&03 after addition of sufficient carrier bicarbonate so that radioact,ive assays could be carried out at infinite thickness. Deproteinization of the reaction mixture for chemical analysis was carried out by adding sufficient trichloroacetic acid to give a final concentration of 5%.
Isolation of Pyruvate and Succinatc For the isolation of succinic acid, the deprot,einized solution was evaporat’ed t,o dryness on a steam cone under mild acid conditions. SWcinic acid was isolated from the residue by the chromatographic procedures described by Busch, Hurlbert, and Potter (7). The free acid was convert,& to it’s sodium salt and recrystallized to con&ant activity from et,hanol. Pyrur-ate was isolated as the 2, &dinitrophenylhydrazone according to the procedures of &Zahler and Huennekerrs (2). Recrystallization to constant. ac*t,ivit,y was carried out from aqueous ethanol solutions. Each of the above products was isolated individually from one of two paired react’ion vessels. In the experiments carried out in the absence of a trapping agent, 40 mg. of succinic acid or lithium pyruvate was added as carrier prior to deproteinizing the reaction mixture. All radioactive determinations were carried out at) infinit#e thickness wit’h Ba,C& obtained by wet oxidation, except the pyruvate 2 ,A-dinitrophenylhydrazones. The lat,ter derivative was cxounted directly but calwlated to the infinite thickness BaC03 basis. The values are expressed ill terms of the per (sent of the total added activity recovered, calcula.ted from the formula: % conversion = pJ1 0’. X S.A. (product) as BaC03 at infinit,e thickness x 100 PM C. X S.A. (substrate) as BaC03 at infinit)e t’hickness RESULTS
AND
DISCUSSION
The abi1it.y of t)he enzyme preparat,ion to oxidize propionat,e-1-W is shown by the data in Table I. Tt can be seen that a,snnwh as 63 % of t>hc
416
JACK
B.
WOLFE
TABLE I of Manometric and Isotopic Data Obtained in the Oxidation of Propionate-i-04 by Washed Rabbit Liver “Particles” Reaction mixture contained: 1 ml. of enzyme preparation, 10 PM propionatel-CY, 5 PM citrate “sparker,” 2 PM adenylic acid, 2 pM MgClz , and KCl-phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml.; incubation 37”, air atmosphere. Comparison
Manometric
Expt.
No.
1 2 3
Oxygen
consumeh PM
5.2 -(Z)
measurements Propionate oxidizedC CM
1.5 1.1 -
Isotopic measurementsa Total added activity recovered Propionate as woe oxidizedC % PM
63 32 28
6.3 3.2 2.8
Time
of incubation min.
120 80 95
0 The total added counts of propionate-l-Cl4 was 5.6 X lo4 counts/min. counted at an infinite, thick layer of BaC03 . b Control flask without propionate subtracted. c Calculations based on oxidation of the substrate to completion.
when
total added activity can be recovered in the respiratory carbon dioxide during a 2-hr. incubation period. In somecasesa negative oxygen uptake was observed where an isotopic measurement of the respiratory carbon dioxide clearly indicated that propionate oxidation had taken place. It is apparent that the oxygen uptake data do not necessarily indicate the degree of substrate oxidation. Table II presents typical data obtained when the enzyme preparation was incubated with propionate-1-CY4, propionate-2-C14, and nn-lactatel-C14. The result’sindicate that D- and n-lactate are very poorly oxidized by these preparations under conditions where propionate is readily metabolized. The apparent lack of an active D- and L-lactate oxidizing system suggestedthat propionate was not oxidized through a metabolic pathway involving D- or L-lactate. The total activity recovered in the respiratory carbon dioxide from both isotopic propionates was approximately equal (Table II). This would be expected if the observed combustion of propionate went toward completion. Effect of Malonate Lehninger (8) has shown that a quantitative conversion of pyruvate to acetoacetate can occur in liver systems poisoned with malonate. Therefore, it would be expected that if propionate was directly oxidized
PROPIONATE
TABLE A Comparison
of Propionate-l-04, by Washed
41;
OXIDATI03
II
Propionate-.%‘XY and m-Lactate-l-C’4 Rabbit Liver “Particles”
Oxidation
The reaction mixture contained: 1 ml. enzyme preparation, 10 FM of the indicated substrate, 5 PM citrate “sparker,” 2 PM adenylic acid, 2 ~crM MgC12 , and KCl-phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml. Incubation was 70 min. at 37” in an air atmosphere. Per cent of totaladded actSty recovered as CWop Substrate
Substrate”
Propionate-1-W Propionate-2-W m-Lactate-l-W
oxidized MY
to GOn
2.6 2.5 0.04
26 25 0.4
a The total added counts of propionate-l-W, propionate-2-04, and m-lactatel-C’4 were 1.55 X 106, 1.45 X 100, and 1.0 X lo7 counts/min., respectively, when counted as BaC03 at infinite thickness.
The E$ect
of Malonate
TABLE III (0.01 M) on Propionate-l-04 by Washed
Rabbit
Liver
and Propionate-d-C14 “Particles”
Oxidation
The reaction mixture contained: 1 ml. enzyme preparation, 10 PM of the indicated substrate, 5 FM citrate “sparker,” 2 PM adenylic acid, 2 PM MgClt , 0.01 M malonate, and KC&phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml. Incubat,ion was for 70 min. at 37” in an air atmosphere. Per cent of total added Propionate-1-W Control plus Control malonate
Respiratory Succinate Pyruvate
COZ
29, 28 0.5, 0.2 0.3, 0.4
0.7, 0.5 3.5, 2.6 0.02, 0.02
activity
recoveredn Propionate-Z-C’4 Control pl US Control malonate
31, 25 -_ -
0.6, 0.4 -
a Values represent two independent experiments. The total added counts of propionate-l-C14 and propionate-2-W were 1.55 X lo6 and 1.45 X lo6 counts/min., respectively, when count,ed as BaCOa at infinite thickness.
by way of an alpha oxidation mechanism, the formation of isotopic carbon dioxide from propionate-l-Cl4 would be less inhibited by malonate than that produced from propionate-2-C14. The effect of malonate on propionate oxidation is presented in Table III. The results show that malonate strongly inhibits the oxidation of propionate, with the isotopic carbon dioxide produced from both propionate-l-Cl4 and propionate-2-V4 being inhibited to approximately the same extent. Although the data suggest that pyruvate is not, a direct intermediate,
to pyruvate
418
JACK
B.
WOLFE
it is not possible to make any definite conclusions since there is evidence that malonate may inhibit at sites other than succinic dehydrogenase (9, 10). Therefore, succinate and pyruvate were isolated from the reaction mixtures in which propionate-l-C4 was oxidized in the presence and absence of malonate. It was found that the presence of malonate greatly reduced the total activity recovered in the isolated pyruvate, whereas the total activity recovered in the succinate was increased (Table III). In fact, a higher total activity was recovered in the succinate than in the respiratory carbon dioxide. It is apparent from the above results, since carboxyl-labeled propionate was used, that malonate is not inhibiting propionate oxidation at the pyruvate level, but rather at the succinate level. The results further suggest that isotopic succinate formation from propionate-l-Cl4 occurs prior to entry of the isotope into pyruvate. Pyruvate and Succinate as Trapping
Agents
The previous experiments employing malonate as an inhibitor provided evidence that succinate was on the direct pathway of propionate oxidation. The total counts recovered in the presence of malonate, however, were not sufficient to conclude that this represented a major metabolic pathway. Consequently, experiments were carried out in which propionate-l-Cl4 was oxidized by the enzyme preparation in the presence of succinate or pyruvate as a trapping agent. It was found that when succinate was employed as the trapping agent, the formation of isotopic carbon dioxide was greatly suppressed (Table IV). Radioactive assays of the succinate isolated from these reaction mixtures gave total activity values approaching that found in the respiratory carbon dioxide of control flasks run in the absence of added succinate. These results indicated that the added nonisotopic succinate is effectively trapping the isotopic succinate formed during the oxidation of propionate-1-CY4, thus diluting the isotope prior to its entry into the respiratory carbon dioxide. The pyruvate trap was far less effective than the succinate in suppressing the formation of isotopic carbon dioxide (Table IV). The respiratory carbon dioxide recovered in these experiments had a higher total activit’y than that found in the isolated pyruvate. These results clearly show that under conditions where an alpha oxidation of propionate is not occurring due to the removal of the soluble enzyme component, “racemase,” succinate rather than pyruvate becomes an obligatory intermediate in a major pathway of propionate
PROPIONSTE TABLE The Effect
of a Pyruvate
OXIDATION
419
IV
or Succinate Trap on the Oxidation Washed Rabbit Liver “Particles”
of Propionate-i-C14
by
The reaction mixture contained: 1 ml. of enzyme preparation, 10 BM of propionate-1-C 14, 5 ~$2 citrate “sparker” (except when succinate was the trapping agent), 2 &U adenylic acid, 2 ELM MgClz , 200 PM of succinate or pyruvate, and KC&phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml. Incubation was for 1 hr. at 37” in an air atmosphere. Per cent of the total Pyruvate Control trap
Respiratory I’yruvatc Succinato
CO2
15.3 0.5
10.6 6.4 -
added
activity
Control
Succinate
17.1, 18.4 0.16,
recovereda
0.22
tral,
0.21, 0.15 12.9, 14.9
a Succinate values represent two independent experiments. The total added counts of propionate-1-W u-as 1.55 X IO8 counts/min. when counted as BaCO$ at, infinite thickness. Calculat,ions based on theoretical amount of pyruvate or succinat,e added.
oxidation. The exact nature of the mechanism involved in the conversion of propionate to succinate is still a matter of conjecture. The data obtained, however, are compatible with a mechanism involving a direct carboxylation of propionate or a derivative to succinat~eas proposed by Lardy (5). A4 similar type of mechanism has been considered for the metabolism of propionate in rumen epithelium tissue (11) and shown t,o exist in microorganisms (12, 13). ACKNOWLEDGMENT The author is grateful for the advice and encouragement,
of Dr. Arne N. Wick.
SUMMARY
l’ropionat,e oxidation by washed rabbit liver “particles” which lack the soluble enzyme component “racemase” was investigated, utilizing radioactive isotopes. It was found that propionate-l-Cl4 and -2-Cl4 was readily oxidized under conditions where m-lactate-l-P was poorly oxidized. The presence of malonate (0.01 M) reduced the isotopic CO,: and pyruvate formed from propionate-l-C14, whereas isotSopiesuccinate formation was increased. During the oxidation of propionate-1-C14, succinatc wasmore effect,ive asa kapping agent than pyruvate. These findings are interpreted as ruling out a direct oxidation of propionate through pyruvate and indicate an alt,ernate pathway of propionate oxidatlion, which has succinate as a direct intermediate.
420
JACK
B. WOLFE
REFERENCES 1. HAHN, A., AND HAARMANN, W., 2. Biol. 90, 231 (1930). 2. MAHLER, H. R., AND HUENNEKENS, F. M., Biochim. et Biophp. Acta 11, 575 (1953). 3. LORBER, V., LIFSON, H., SAKAMI, W., AND WOOD, H. G., J. Biol. Chem. 183, 531 (1950). 4. SHREEVE, W. W., J. Biol. Chem. 196, 1 (1952). 5. LARDY, H. A., Proc. Natl. Acad. Sci. U. S. 38, 1003 (1952). 6. U~REIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques and Related Methods for the Study of Tissue Metabolism”. Burgess Publ. Co., Minneapolis, 1946. 7. BUSCH, H., HURLBERT, R. B., AND POTTER, V. R., J. BioZ. Chem. 188, 717 (1952). 8. LEHNINGER, A. L., J. Biol. Chem. 184,291 (1946). 9. PARDEE, A. B., AND POTTER, V. R., J. Biol. Chem. 178,241 (1949). 10. WEINHOUSE, S., MILLINGTON, R. H., AND FRIEDMAN, B., J. Biol. Chcm. 181, 489 (1949). 11. PENNINGTON, R. J., Biochem. J. 68,410 (1954). 12. DELWICHE, E. A., PHARES, E. F., AND CARSON, S. F., Federation Proc. 13, 659 (1953). 13. WHITELY, H. R., Proc. Natl. Acad. Sci. U. S. 39, 781 (1953).
Oxidation
by Washed Rabbit Liver “Particles” Jack B. Wolfe’
From the Scripps Metabolic Received
Clinic, La Jolla, California
February
5, 1955
INTRODUCTION
The metabolic pathway of propionate oxidation in mammalian systems still remains to be elucidated. One possible pathway for the utilization of this compound is a direct oxidation to pyruvate. This mechanism was initially proposed for animal tissuesby Hahn and Haarmann (1) who noted an increase in pyruvate formation when propionate was incubated with muscle mince. Mahler and Huennekens (2) using a rabbit liver “cyclophorase” system also provided evidence for a modified alpha oxidation mechanism. These latter authors proposed the reaction sequence: propionate, acrylate, L-lactate, n-lactate, pyruvate, etc., to account for their experimental data. An alternate pathway in which propionate is converted into the J-carbon dicarboxylic acids by a mechanism not involving pyruvate has been suggested by isotopic experiments (3, 4). Lardy (5) considers this pathway to involve a carboxylation reaction with the formation of succinate. In the present study isotopic tracers were utilized to investigate the pathways involved in the oxidation of propionate by washed rabbit liver “particles.” METHODS
Preparation
of Washed
“Particles”
Twenty grams of freshly removed rabbit liver was minced into a Waring blendor containing 80 ml. of cold KCl-phosphate buffer solution (6 parts 0.85% KC1 to 1 part 0.1 2M Na2HP04 , pH 7.5). The mixture was blended for 30 sec. at one-half maximum speed, filtered through gauze, and centrifuged in the cold (4 f 1”) for 5 min. at 4500 X 9. The resulting particles were washed two times with 80 ml. of cold KCl-phosphate buffer solution and centrifuged at 4500 X g for 4 min. after 1 Charles
Willard
Stimsom
Postdoctoral
Fellow 414
in Biochemistry
PROPIONSTE
OXIDATION
each washing. The washed “residue” was finally suspended in a volume of cold KCI-phosphate buffer solution so that 1 ml. of the suspension represented the residue obtained from 0.66 g. of the original weight of the liver. Incubations were conducted in Warburg vessels at 37” in an air atmosphere. Oxygen ronsumption was measured according to conventional manometric procedures (6). At the end of the incubation period 50% sulfuric acid was added to stop the reaction and to release carbon dioxide from the reaction mixture. The respiratory carbon dioxide was collected in alkali and then precipitated as B&03 after addition of sufficient carrier bicarbonate so that radioact,ive assays could be carried out at infinite thickness. Deproteinization of the reaction mixture for chemical analysis was carried out by adding sufficient trichloroacetic acid to give a final concentration of 5%.
Isolation of Pyruvate and Succinatc For the isolation of succinic acid, the deprot,einized solution was evaporat’ed t,o dryness on a steam cone under mild acid conditions. SWcinic acid was isolated from the residue by the chromatographic procedures described by Busch, Hurlbert, and Potter (7). The free acid was convert,& to it’s sodium salt and recrystallized to con&ant activity from et,hanol. Pyrur-ate was isolated as the 2, &dinitrophenylhydrazone according to the procedures of &Zahler and Huennekerrs (2). Recrystallization to constant. ac*t,ivit,y was carried out from aqueous ethanol solutions. Each of the above products was isolated individually from one of two paired react’ion vessels. In the experiments carried out in the absence of a trapping agent, 40 mg. of succinic acid or lithium pyruvate was added as carrier prior to deproteinizing the reaction mixture. All radioactive determinations were carried out at) infinit#e thickness wit’h Ba,C& obtained by wet oxidation, except the pyruvate 2 ,A-dinitrophenylhydrazones. The lat,ter derivative was cxounted directly but calwlated to the infinite thickness BaC03 basis. The values are expressed ill terms of the per (sent of the total added activity recovered, calcula.ted from the formula: % conversion = pJ1 0’. X S.A. (product) as BaC03 at infinit,e thickness x 100 PM C. X S.A. (substrate) as BaC03 at infinit)e t’hickness RESULTS
AND
DISCUSSION
The abi1it.y of t)he enzyme preparat,ion to oxidize propionat,e-1-W is shown by the data in Table I. Tt can be seen that a,snnwh as 63 % of t>hc
416
JACK
B.
WOLFE
TABLE I of Manometric and Isotopic Data Obtained in the Oxidation of Propionate-i-04 by Washed Rabbit Liver “Particles” Reaction mixture contained: 1 ml. of enzyme preparation, 10 PM propionatel-CY, 5 PM citrate “sparker,” 2 PM adenylic acid, 2 pM MgClz , and KCl-phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml.; incubation 37”, air atmosphere. Comparison
Manometric
Expt.
No.
1 2 3
Oxygen
consumeh PM
5.2 -(Z)
measurements Propionate oxidizedC CM
1.5 1.1 -
Isotopic measurementsa Total added activity recovered Propionate as woe oxidizedC % PM
63 32 28
6.3 3.2 2.8
Time
of incubation min.
120 80 95
0 The total added counts of propionate-l-Cl4 was 5.6 X lo4 counts/min. counted at an infinite, thick layer of BaC03 . b Control flask without propionate subtracted. c Calculations based on oxidation of the substrate to completion.
when
total added activity can be recovered in the respiratory carbon dioxide during a 2-hr. incubation period. In somecasesa negative oxygen uptake was observed where an isotopic measurement of the respiratory carbon dioxide clearly indicated that propionate oxidation had taken place. It is apparent that the oxygen uptake data do not necessarily indicate the degree of substrate oxidation. Table II presents typical data obtained when the enzyme preparation was incubated with propionate-1-CY4, propionate-2-C14, and nn-lactatel-C14. The result’sindicate that D- and n-lactate are very poorly oxidized by these preparations under conditions where propionate is readily metabolized. The apparent lack of an active D- and L-lactate oxidizing system suggestedthat propionate was not oxidized through a metabolic pathway involving D- or L-lactate. The total activity recovered in the respiratory carbon dioxide from both isotopic propionates was approximately equal (Table II). This would be expected if the observed combustion of propionate went toward completion. Effect of Malonate Lehninger (8) has shown that a quantitative conversion of pyruvate to acetoacetate can occur in liver systems poisoned with malonate. Therefore, it would be expected that if propionate was directly oxidized
PROPIONATE
TABLE A Comparison
of Propionate-l-04, by Washed
41;
OXIDATI03
II
Propionate-.%‘XY and m-Lactate-l-C’4 Rabbit Liver “Particles”
Oxidation
The reaction mixture contained: 1 ml. enzyme preparation, 10 FM of the indicated substrate, 5 PM citrate “sparker,” 2 PM adenylic acid, 2 ~crM MgC12 , and KCl-phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml. Incubation was 70 min. at 37” in an air atmosphere. Per cent of totaladded actSty recovered as CWop Substrate
Substrate”
Propionate-1-W Propionate-2-W m-Lactate-l-W
oxidized MY
to GOn
2.6 2.5 0.04
26 25 0.4
a The total added counts of propionate-l-W, propionate-2-04, and m-lactatel-C’4 were 1.55 X 106, 1.45 X 100, and 1.0 X lo7 counts/min., respectively, when counted as BaC03 at infinite thickness.
The E$ect
of Malonate
TABLE III (0.01 M) on Propionate-l-04 by Washed
Rabbit
Liver
and Propionate-d-C14 “Particles”
Oxidation
The reaction mixture contained: 1 ml. enzyme preparation, 10 PM of the indicated substrate, 5 FM citrate “sparker,” 2 PM adenylic acid, 2 PM MgClt , 0.01 M malonate, and KC&phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml. Incubat,ion was for 70 min. at 37” in an air atmosphere. Per cent of total added Propionate-1-W Control plus Control malonate
Respiratory Succinate Pyruvate
COZ
29, 28 0.5, 0.2 0.3, 0.4
0.7, 0.5 3.5, 2.6 0.02, 0.02
activity
recoveredn Propionate-Z-C’4 Control pl US Control malonate
31, 25 -_ -
0.6, 0.4 -
a Values represent two independent experiments. The total added counts of propionate-l-C14 and propionate-2-W were 1.55 X lo6 and 1.45 X lo6 counts/min., respectively, when count,ed as BaCOa at infinite thickness.
by way of an alpha oxidation mechanism, the formation of isotopic carbon dioxide from propionate-l-Cl4 would be less inhibited by malonate than that produced from propionate-2-C14. The effect of malonate on propionate oxidation is presented in Table III. The results show that malonate strongly inhibits the oxidation of propionate, with the isotopic carbon dioxide produced from both propionate-l-Cl4 and propionate-2-V4 being inhibited to approximately the same extent. Although the data suggest that pyruvate is not, a direct intermediate,
to pyruvate
418
JACK
B.
WOLFE
it is not possible to make any definite conclusions since there is evidence that malonate may inhibit at sites other than succinic dehydrogenase (9, 10). Therefore, succinate and pyruvate were isolated from the reaction mixtures in which propionate-l-C4 was oxidized in the presence and absence of malonate. It was found that the presence of malonate greatly reduced the total activity recovered in the isolated pyruvate, whereas the total activity recovered in the succinate was increased (Table III). In fact, a higher total activity was recovered in the succinate than in the respiratory carbon dioxide. It is apparent from the above results, since carboxyl-labeled propionate was used, that malonate is not inhibiting propionate oxidation at the pyruvate level, but rather at the succinate level. The results further suggest that isotopic succinate formation from propionate-l-Cl4 occurs prior to entry of the isotope into pyruvate. Pyruvate and Succinate as Trapping
Agents
The previous experiments employing malonate as an inhibitor provided evidence that succinate was on the direct pathway of propionate oxidation. The total counts recovered in the presence of malonate, however, were not sufficient to conclude that this represented a major metabolic pathway. Consequently, experiments were carried out in which propionate-l-Cl4 was oxidized by the enzyme preparation in the presence of succinate or pyruvate as a trapping agent. It was found that when succinate was employed as the trapping agent, the formation of isotopic carbon dioxide was greatly suppressed (Table IV). Radioactive assays of the succinate isolated from these reaction mixtures gave total activity values approaching that found in the respiratory carbon dioxide of control flasks run in the absence of added succinate. These results indicated that the added nonisotopic succinate is effectively trapping the isotopic succinate formed during the oxidation of propionate-1-CY4, thus diluting the isotope prior to its entry into the respiratory carbon dioxide. The pyruvate trap was far less effective than the succinate in suppressing the formation of isotopic carbon dioxide (Table IV). The respiratory carbon dioxide recovered in these experiments had a higher total activit’y than that found in the isolated pyruvate. These results clearly show that under conditions where an alpha oxidation of propionate is not occurring due to the removal of the soluble enzyme component, “racemase,” succinate rather than pyruvate becomes an obligatory intermediate in a major pathway of propionate
PROPIONSTE TABLE The Effect
of a Pyruvate
OXIDATION
419
IV
or Succinate Trap on the Oxidation Washed Rabbit Liver “Particles”
of Propionate-i-C14
by
The reaction mixture contained: 1 ml. of enzyme preparation, 10 BM of propionate-1-C 14, 5 ~$2 citrate “sparker” (except when succinate was the trapping agent), 2 &U adenylic acid, 2 ELM MgClz , 200 PM of succinate or pyruvate, and KC&phosphate buffer solution (pH 7.5) to a total volume of 2.6 ml. Incubation was for 1 hr. at 37” in an air atmosphere. Per cent of the total Pyruvate Control trap
Respiratory I’yruvatc Succinato
CO2
15.3 0.5
10.6 6.4 -
added
activity
Control
Succinate
17.1, 18.4 0.16,
recovereda
0.22
tral,
0.21, 0.15 12.9, 14.9
a Succinate values represent two independent experiments. The total added counts of propionate-1-W u-as 1.55 X IO8 counts/min. when counted as BaCO$ at, infinite thickness. Calculat,ions based on theoretical amount of pyruvate or succinat,e added.
oxidation. The exact nature of the mechanism involved in the conversion of propionate to succinate is still a matter of conjecture. The data obtained, however, are compatible with a mechanism involving a direct carboxylation of propionate or a derivative to succinat~eas proposed by Lardy (5). A4 similar type of mechanism has been considered for the metabolism of propionate in rumen epithelium tissue (11) and shown t,o exist in microorganisms (12, 13). ACKNOWLEDGMENT The author is grateful for the advice and encouragement,
of Dr. Arne N. Wick.
SUMMARY
l’ropionat,e oxidation by washed rabbit liver “particles” which lack the soluble enzyme component “racemase” was investigated, utilizing radioactive isotopes. It was found that propionate-l-Cl4 and -2-Cl4 was readily oxidized under conditions where m-lactate-l-P was poorly oxidized. The presence of malonate (0.01 M) reduced the isotopic CO,: and pyruvate formed from propionate-l-C14, whereas isotSopiesuccinate formation was increased. During the oxidation of propionate-1-C14, succinatc wasmore effect,ive asa kapping agent than pyruvate. These findings are interpreted as ruling out a direct oxidation of propionate through pyruvate and indicate an alt,ernate pathway of propionate oxidatlion, which has succinate as a direct intermediate.
420
JACK
B. WOLFE
REFERENCES 1. HAHN, A., AND HAARMANN, W., 2. Biol. 90, 231 (1930). 2. MAHLER, H. R., AND HUENNEKENS, F. M., Biochim. et Biophp. Acta 11, 575 (1953). 3. LORBER, V., LIFSON, H., SAKAMI, W., AND WOOD, H. G., J. Biol. Chem. 183, 531 (1950). 4. SHREEVE, W. W., J. Biol. Chem. 196, 1 (1952). 5. LARDY, H. A., Proc. Natl. Acad. Sci. U. S. 38, 1003 (1952). 6. U~REIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques and Related Methods for the Study of Tissue Metabolism”. Burgess Publ. Co., Minneapolis, 1946. 7. BUSCH, H., HURLBERT, R. B., AND POTTER, V. R., J. BioZ. Chem. 188, 717 (1952). 8. LEHNINGER, A. L., J. Biol. Chem. 184,291 (1946). 9. PARDEE, A. B., AND POTTER, V. R., J. Biol. Chem. 178,241 (1949). 10. WEINHOUSE, S., MILLINGTON, R. H., AND FRIEDMAN, B., J. Biol. Chcm. 181, 489 (1949). 11. PENNINGTON, R. J., Biochem. J. 68,410 (1954). 12. DELWICHE, E. A., PHARES, E. F., AND CARSON, S. F., Federation Proc. 13, 659 (1953). 13. WHITELY, H. R., Proc. Natl. Acad. Sci. U. S. 39, 781 (1953).