Phosphorylation associated with succinate decarboxylation to propionate in Ascaris mitochondria

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 202, No. 2, July, pp. 388-395, 1980

Phosphorylation Associated with Succinate Decarboxylation to Propionate in Ascaris Mitochondrial HOWARD Department of Biology,

J. SAZ AND SUSAN of Notre

University

M. PIETRZAK

Dame,

Notre

Dame,

Indiana 46556

Received February 7, 1980 In mammalian tissues, propionyl CoA carboxylase and methylmalonyl CoA mutase act physiologically primarily in the ATP requiring direction of succinate formation. Therefore, propionate is glycogenic. However, many invertebrates and bacteria accumulate propionate from succinate. Employing acyl CoA transferase, propionyl CoA carboxylase, and methylmalonyl CoA mutase in the direction of propionate formation, substrate level ATP should be generated. The intestinal worm, Ascaris lumbricoides possesses mitochondria which function anaerobically and accumulate propionate and volatile fatty acids derived from propionate. Results of the present study indicate that a site of mitochondrial phosporylation may be present at the substrate level during the decarboxylation of succinate to propionate and COz. Mitochondrial preparations from Ascaris muscle exhibit propionyl CoA carboxylase, methylmalonyl CoA mutase, and acyl CoA transferase activities. Inorganic 32P is esterified during succinate decarboxylation. In accord with the proposed reactions, both succinate decarboxylation and 32P esterification are stimulated six- to eightfold upon the addition of propionyl CoA, and both stimulations are inhibited by avidin.

In mammalian tissues, propionate is glycogenic as a consequence of its conversion to succinate catalyzed by propionyl CO, + ATP + E (propionyl

COA carboxylase)

“COS” - E + propionyl methylmalonyl

CoA carboxylase and methylmalonyl CoA mutase. The reactions of this sequence, as elucidated by Kaziro and Ochoa (l), may be illustrated as follows:

CoA c methylmalonyl

mutase + vitamin CoA k

succinyl CoA + propionate CO, + ATP + propionate For these reactions to proceed, the presence of methylmalonyl CoA racemase is required, but has been omitted, since it would not affect the overall balance. Under appropriate physiological conditions all of these reactions could be reversible. That is, the decarboxylation of succinate might give rise to both propionate and ATP. Many of the bacteria, parasitic helminths, and other invertebrates form propionate as a major fermentation product (2-5), but the possibility of obtaining 0003-9861/80/080388-08$02.00/O Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

= “COZ)) - E + ADP + Pi,

B,, coenzyme

* propionyl

CoA + E, ’ succinyl CoA,

CoA + succinate.

e succinate + ADP + Pi.

111 VI [31 [41

Sum [l-4]

energy from the conversion of succinate to propionate has not been fully examined. Tkachuck et al. (6) reported that the adult tapeworm, Spirometra mansonoides , accumulated propionate, contained high activities of propionyl CoA carboxylase and methylmalonyl CoA isomerase, and incor* This investigation was supported in part by Grants AI-09483, AI-10512, and AI-07030 from the National Institutes of Health, United States Public Health Service. 388

PHOSPHORYLATIONSUCCINATE

DECARBOXYLATION

porated 32Pi into ATP upon incubation of methylmalonyl CoA with crude extracts of S. mansonoides. The presence of the carboxylase and mutase in the propionateforming liver fluke, Fasciola hepatica, was reported (7), and subsequently, indirect evidence indicated that phosphorylation may be associated with succinate decarboxylation in this trematode (8). Of particular interest are the recent reports of Meyer and Meyer (9) and Meyer et al. (10). These authors purified the acyl CoA carboxylases from the free living nematode Turbatrix aceti and the cestode Spirometra mansonoides, respectively, and in both cases activity on propionyl CoA was considerably greater than that on acetyl or butyryl CoA. Mitochondria from the intestinal roundworm, Ascaris lumbricoides var. suum, function anaerobically and accumulate succinate and propionate (11). Propionate then serves as a precursor for two of the major products ofAscaris carbohydrate fermentation, 2-methylvalerate and 2-methylbutyrate. Preliminary evidence has been presented indicating that all of the reactions listed above occur in Ascaris and that phosphorylation is associated with the decarboxylation of succinate to propionate (12). MATERIALS

AND METHODS

Adult female Ascaris lumbricoides var. suum were obtained from the slaughterhouse and mitochondria were prepared as previously described (13) from dissected female muscle strips employing a medium composed of 0.24 M sucrose, 5 ITIM EDTA, and 0.15% crystalline bovine serum albumin, pH = 7.4. Where indicated, sonicated mitochondria were prepared from washed mitochondria obtained from 10 g (wet weight) of muscle. The washed mitochondria were made to 3.0 ml with mitochondrial medium and sonicated in the cold by eight pulses of 15 s each with 30-s cooling intervals in a Heat Systems sonicator Model W 185 fitted with a microtip at a power setting of 50 W. The mitochondrial soluble fraction was prepared from sonicated mitochondria by centrifugation at 269,000 g for 30 min. Where indicated, sonicated mitochondria were dialyzed at 4°C for 12 h against 500 vol of 0.02 M Tris-HCl containing 0.004 M potassium phosphate, pH = 7.4. Succinate decarboxylation was assayed by determining the radioactivity recovered in the CO* or propionate formed from [1,4-‘4C]succinate or [2,3-14C]-

IN Ascaris

MITOCHONDRIA

389

succinate, respectively. To determine radioactivity in propionate, the coenzyme A ester was hydrolyzed by the addition of KOH to a pH of 9 and heated at 70°C for 10 min. Under these conditions, less than 1% of the coenzyme A ester remained, as determined experimentally employing the known esters. The mixture then was acidified and distilled almost to dryness. The distillate was collected in a flask immersed in liquid nitrogen, neutralized, concentrated, and subjected to gas-liquid chromatography as previously described (14). Propionic acid was recovered from the column by trapping in alkali and its content of radioactivity determined. Propionyl CoA carboxylase activity was determined by measuring the incorporation of NaH’VO, into the nonvolatile fraction (15). Methylmalonyl CoA mutase was assayed according to Tkachuck et al. (6) employing [*4C]methylmalonyl CoA and isolating [14C]succinate at the termination of incubation by chromatography on DEAE Sephadex columns. Acyl CoA transferase was determined by incubating an acyl CoA donor (succinyl CoA or propionyl CoA) with an acceptor (propionate, succinate, or 2methylbutyrate). Details of incubations are listed in the legend to the appropriate figures. After incubation at 30°C for 20 min, reactions were terminated by the addition of 0.2 ml of 12% perchloric acid. Mixtures were then neutralized with 1.0 M KOH and centrifuged. Supernatants were absorbed onto washed Sep-Pak Cn, cartridges (Waters Associates, Inc., Milford, Mass.). Compounds of high polarity were removed by eluting with H,O. Acyl CoA esters then were eluted with 50% methanol. Aliquots containing the acyl CoA esters were submitted directly to separation by means of paired ion hplc* on a PBondapak C,, column, 4 mm i.d. x 30 cm. Separations were accomplished by elution with 43% methanol in water containing PIC A reagent (Waters Associates). The flow rate was 2.0 mYmin and compounds were detected by light absorbtion at 250 nm. Retention times were characteristic and reproducible for both known acyl CoA esters and reaction mixtures with or without added known CoA derivatives. CoA derivatives of the following acids were employed as standards: succinic, acetic, propionic, butyric, n-Valerie, and tiglic. Where indicated, peaks eluting were collected and spectra were determined employing a Model 25 Beckman spectrophotometer. Phosphorylation was assayed by determining the incorporation of radioactive inorganic orthophosphate (32P) into the organic fraction, essentially according to the procedure of Grunberg-Manago et al. (16). Protein was determined according to Lowry et al. (17). All experiments reported were repeated a minimum of five times, employing ascarids obtained at different * Abbreviation chromatography.

used: hplc, high-pressure

liquid

SAZ AND PIETRZAK

390

times. Although absolute levels of activity differed with each preparation, results were qualitatively similar in all instances. Acyl CoA derivatives were purchased from P-L Biochemicals, Milwaukee, Wisconsin. Methanol employed for HPLC was purchased from Burdick and Jackson Laboratories, Inc., Muskegon, Michigan. [14C]Methylmalonyl CoA, [1,4-“Clsuccinate, [2,3-“‘Clsuccinate, and NaH14C0, were obtained from New England Nuclear Corporation, Boston, Massachusetts. Vitamin B,, coenzyme and avidin were purchased from Sigma Chemical Company, St. Louis, Missouri.

TABLE

DECARBOXYLATION 0F [2,3-W]Succ1mT~ Am [1,4-14C]S~~~~~A~~ BY Ascaris MUSCLE MITOCHONDRIA~ Product recovered* (nmol/20 min/mg protein) Experiments 1

RESULTS

Succinate decarboxylation by Ascaris mitochondria. Ascark muscle strips have

been shown previously to catalyze the reversible decarboxylation of succinate (11). Mitochondrial preparations demonstrate the same capability (Table I). [14C]Propionate and 14C02 are recovered when [2,3J4C]succinate and [1,4-14C]succinate, respectively, are employed as the anaerobic substrates. Most interesting is the approximately &fold stimulation of activity observed upon the addition of propionyl CoA. In 10 repeats of this experiment (5 employing [2,3J4C]succinate and 5 using [1,4-14C]succinate), this stimulation by propionyl CoA was always observed and ranged from 3.9- to 5.6-fold. As noted in this experiment, stimulations by ATP and CoA were consistently less than formed with propionyl CoA. Substitution of CoA plus ATP for propionyl CoA resulted in an approximate 2-fold stimulation, while ATP itself stimulated succinate decarboxylation only slightly. The mechanism of this stimulation by propionyl CoA could be predicted on the basis of reactions [l] through [4]. Propionyl CoA would be required for the activation of succinate via transacylation and would be regenerated in the reaction. It is of interest that avidin, an inhibitor of the biotin requiring propionyl CoA carboxylase, had a marked inhibitory effect upon this succinate decarboxylation system, in 5 experiments inhibiting the propionyl CoA stimulation from ‘79 to 100%. Propionyl CoA carboxylase, methylmalonyl CoA mutase, and acyl CoA transferase activities of Ascaris muscle. Pro-

pionyl

CoA carboxylase

activity

can be

I

2

Additions

‘WO*

Zero time [2,3-*4C]Succinate + propionyl CoA [2,3-“C]Succinate Zero time [1,4-14C]Succinate + propionyl CoA [1,4-L4C]Succinate [1,4-L4C]Succinate + CoA + ATP [1,4-‘4C]Succinate + ATP [1,4-“‘C]Succinate + propionyl CoA + avidin

[‘4C]Propionate 4 62 15

4 64 12 22 16 23

u In experiments 1 and 2, dialyzed sonicated mitochondrial preparations were employed as the source of enzymes. Each vessel contained 2.5 mgprotein. Where indicated, avidin concentration was 0.05 mg/ml. Incubations were performed in a Warburg respirometer under an atmosphere of nitrogen at 30°C in a total volume of 1.0 ml. In addition to enzyme, each vessel contained in pmol: Tris-HCl, pH = 7.4, 40; ADP, 2; MgCI,, 10; potassium phosphate, pH = 7.4, 12; coenzyme B12, 2; reduced glutathione, 5; KCl, 20; 1,4[14C]succinate or 2,3[14C]succinate where indicated, 10, (85,750 and 112,300 dpm/pmol, respectively). Where indicated, each of the following were added: propionyl CoA, 2; coenzyme A, lithium salt, 2; ATP, 2. Reactions were terminated after 20 min by acidification with 0.11 ml of 40% trichloroacetic acid. Where indicated, CF02 was collected in hyamine hydroxide. * Zero time control values were obtained employing vessels which contained both radioactive suecinate and propionyl CoA.

demonstrated in mitochondrial fractions of muscle as determined by the incorporation of NaH14C03 into the acyl CoA substrate (Table II). ATP and magnesium are required for optimal activity and, as previously reported by Meyer et al. (10) for what appears to be a similar enzyme

Ascaris

PHOSPHORYLATION/SUCCINATE TABLE

DECARBOXYLATION

II

PROPIONYL COA CARBOXYLASE ACTIVITY OF Ascaris MUSCLE PREPARATIONS’ System

Propionyl CoA carboxylase activity0

Complete -ATP - KC1 (NaATP) -KC1 (KATP) - MgCl, Acetyl CoA substrate Butyryl CoA substrate Valery1 CoA substrate Tiglyl CoA substrate -ATP + GTP -ATP + CTP -ATP + UTP

20.3 0.3 2.4 10.3 1.0 5.3 5.7 3.2 4.1 2.1 2.3 1.3

n Sonicated mitochondria were used as the source of enzyme, and each vessel contained 1.4 mg of protein. Where indicated, vessels contained 4 pmol of the indicated nucleotide and 1 pmol of the indicated acyl CoA ester. Vessels were incubated in air at 37°C in a total volume of 1.0 ml. In addition to enzyme the complete system contained in pmol: Tris-HCl, pH = 7.4, 116; propionyl CoA, 1; MgCl,, 12; reduced glutathione, 5; sodium ATP, 4; KCl, 20; [14C]sodium bicarbonate, 10 (70,600 dpm/pmol). After 15 min of incubation, vessels were cooled in ice and 0.4 ml of 20% trichloroacetic acid was added. Nitrogen was bubbled through this solution for 5 min to release residual CO, prior to counting radioactivity. b Nanomoles of NaH14C0, incorporated into the nonvolatile fraction/min/mg protein.

from Spirometra mansonoides, the Ascaris propionyl CoA carboxylase required K+ for optimal activity. Acyl CoA derivatives other than propionyl CoA also stimulated the incorporation of 14C02, but at a considerably slower rate. As with other propionyl CoA carboxylases, the Ascaris enzyme is specific for ATP. Nucleotides GTP, CTP, and UTP are essentially without activity. The very low levels of activity reported could be accounted for by small contamination of samples with ATP. Incubation of [ 14C]methylmalonyl CoA with either Ascaris cytosol or mitochondrial preparations resulted in the accumulation of [14C]succinate demonstrating the presence of methylmalonyl CoA mutase (Table III). The enzyme was stable to dialysis.

391

IN Ascaris MITOCHONDRIA

However, even after dialysis, the addition of vitamin B,, coenzyme had only a small stimulatory effect upon the reaction. This suggests a tight binding between the mutase and the vitamin B12 coenzyme which appears not to be removed by dialysis. Several acyl CoA transferase activities were demonstrable in Ascaris mitochondria. A dialyzed soluble preparation from Ascaris muscle mitochondria was incubated with succinyl CoA as the CoA donor and propionate as the CoA acceptor at a pH = 8.1 (Fig. 1). One vessel, to which acid was added prior to the enzyme, served as a zero time control (Fig. 1, Curve A). The second vessel was incubated for 20 min prior to the addition of acid (Fig. 1, Curve B); coenzyme A derivatives in each flask then were separated by means of paired ion hplc. In the zero time control flask, one quantiTABLE

III

METHYLMALONYL CoA MUTASE ACTIVITY IN Ascaris MUSCLE PREPARATIONS’ Experiment

Preparation

Methylmalonyl CoA mutase activityh

1

Sonicated mitochondria cytoso1

45.5 22.5

2

Dialyzed sonicated mitochondria Dialyzed sonicated mitochondria minus B,,

78.5 67.0

a All vessels contained 0.18 mg of protein derived from the indicated source. Incubations were in air at 37°C in a total volume of 0.1 ml. In addition to enzyme each vessel contained in pmol: potassium phosphate, pH = 7.4, 8; coenzyme B,,, 2. Vessels were preincubated 10 min prior to the addition of 0.21 pmol of [‘4C]methylmalonyl CoA (210,000 dpml pmol). Vessels were incubated an additional 20 min. Reactions were stopped and the CoA esters were hydrolyzed by heating in alkali as described in the text. After neutralizing, an appropriate aliquot from each vessel was applied to a DEAE Sephadex column (3.5 x 0.5 cm). Propionate, succinate, and methylmalonate were eluted separately with 5 ml each of 0.01 M, 0.05 M, and 0.20 M formic acid, respectively, and the radioactivity was determined. b Nanomoles of [‘4C]methylmalonyl CoA converted to succinateiminlmg protein.

392

SAZ AND PIETRZAK

tatively major peak was obtained which corresponded in retention time to the substrate of the reaction, succinyl CoA (Fig. 1, Curve A, peak 1). It is of interest that a small, but significant, endogenous peak also was present which corresponded to known propionyl CoA (peak 2). After incubation for 20 min, a different profile was obtained (Fig. 1, Curve B, peaks 1 and 2). The suc-

LL A

I I

0

2

I

2

C

2

D

C

I \

/’ 2 I

I-

i

IJ

FIG. 1. Acyl CoA transferase activity (succinyl CoA donating to propionate) in dialyzed, soluble preparations from Ascoti muscle mitochondria. (A) High-pressure liquid chromatogram of acyl CoA esters from the zero time control; (B) chromatogram after 20 min incubation; (C) cochromatography of known succinyl CoA with an aliquot from the 20min incubation; (D) cochromatography of propionyl CoA with an aliquot from the 20-min incubation. In addition to 1.1 mg of protein, each flask contained the following in micromoles and in a final volume of 0.65 ml: Tris-HCl, pH = 8.1, 35; MgCI,, 2.5; succinyl CoA, 0.24; sodium propionate, 20. Vessels were incubated at 30°C for 20 min. Reactions were terminated by the addition of 0.2 ml of 12% perchloric acid. Vessels were incubated identically, except that the acid was added to the zero time control prior to the addition ofthe enzyme. Samples were prepared for hplc and chromatographed as described in the text. Peaks labeled 1 and 2 correspond to succinyl CoA and propionyl CoA, respectively.

D

I 2

,iL FIG. 2. Acyl CoA transferase activity (propionyl CoA donating to succinate) in dialyzed, soluble preparations from Ascaris muscle mitochondria. (A) Highpressure liquid chromatogram of acyl CoA esters from the zero time control; (B) chromatogram after incubation for 20 min at pH = 7.0 (C) chromatogram after incubation for 20 min at pH = 6.5; (D) cochromatography of succinyl CoA with an aliquot from the 26min incubation. Conditions of incubation were as outlined in the legend to Fig. 1, except that phosphate buffers of the indicated pH values were employed and propionyl CoA (0.24 -01) and sodium succinate (20 pmol) were employed as acyl donor and acceptor, respectively. Peaks labeled 1 and 2 correspond to succinyl CoA and propionyl CoA, respectively.

cinyl CoA peak was much smaller, while the propionyl CoA peak was increased to become the major acyl CoA component present. Quantitation by peak height measurements indicated a disappearance of 137 nmol of succinyl CoA and a concomitant appearance of 73 nmol of propionyl CoA. The probable formation of other metabolic products from succiny1 CoA in these crude preparations was not examined further. To further substantiate the identity of the peaks obtained on hplc, two aliquots of the

PHOSPHORYLATION/SUCCINATE

DECARBOXYLATION

L A

2 I

FIG. 3. Acyl CoA transferase activity (succinyl CoA donating to Zmethylbutyrate) in dialyzed soluble preparations from Ascuris muscle mitochondria. (A) High-pressure liquid chromatogram of acyl CoA esters from the zero time control; (B) chromatogram after incubation for 20 min. Conditions of incubation were as described in the legend to Fig. 1, except that sodium 2-methylbutyrate (20 pmol) was the acceptor.

20-min incubation mixture (Fig. 1, Curve B) were taken and cochromatographed with known succinyl CoA (Fig. 1, Curve C) and known propionyl CoA (Fig. 1, Curve D), respectively. As indicated, succinyl CoA cochromatographed with peak 1 and propionyl CoA cochromatographed with peak 2. These findings are in accord with the presence of an acyl CoA transferase in Ascaris mitochondria which catalyzes the transfer of CoA from succinyl CoA to propionate. Initial attempts to demonstrate the reverse reaction which would be required physiologically, i.e., the transfer of CoA from propionyl CoA to succinate were negative. At a pH of 8.1 essentially no reaction occurred. However, in a more acid environment, the reaction was demonstrated readily (Fig. 2). The zero time control exhibited only one major peak which corresponded to the substrate, propionyl CoA

IN Ascaris MITOCHONDRIA

393

(Fig. 2, Curve A, peak 2). Incubation for 20 min at pH = 7.0 resulted in the separation of two acyl CoA derivatives, succinyl CoA, and a decreased amount of propionyl CoA (Fig. 2, Curve B, peaks 1 and 2, respectively). Enzymatic activity was dramatically higher at pH = 6.5 (Fig. 2, Curve C). At this lower pH, almost all of the propionyl CoA was replaced by succinyl CoA. The addition of known succinyl CoA to an aliquot of the incubation mixture which gave rise to Fig. 2, Curve C, indicated that succinyl CoA cochromatographed with peak 1, as would be expected (Curve D). To ascertain further that peak 1 of Curve C (Fig. 2) was indeed an acyl CoA derivative, the peak was collected from the chromatography apparatus and its spectrum compared with that of known succinyl CoA. Both the biological sample and the known succinyl CoA possessed the same absorbtion maximum at 260 nm, which would result from the CoA moiety. These findings indicate the presence of an acyl CoA transferase in Ascaris muscle mitochondria which catalyzes the transfer of CoA from propionyl CoA to succinate. This would be the direction of the reaction required for the proposed physiological function of the enzyme. Propionyl CoA serves as an intermediate in the synthesis of 2-methylbutyrate in Ascaris muscle (11, 15). The possibility of conserving some of the energy involved in the synthesis of this branched-chain acid exists, since it appears that acyl CoA transferase activity is present which catalyzes the transfer of CoA from succinyl CoA to 2-methylbutyrate, forming 2-methylbutyryl CoA (Fig. 3). Succinyl CoA was the major peak recovered from the zero time control flask (Fig. 3, Curve A, peak 1). Incubation for 20 min resulted in a decrease in the succinyl CoA peak and the appearance of a new peak (peak 2), which corresponded in retention time to the CoA ester of a C, acid (e.g., n-Valery1 CoA), and presumably was 2-methylbutyryl CoA. Although the retention time of known 2-methylbutyryl CoA in this system remains to be determined, the biologically formed compound was collected, hydrolyzed with KOH, and steam distilled after acidification. The dis-

394

SAZ AND PIETRZAK

tillate was concentrated and subjected to gas chromatography (14). A peak was obtained which cochromatographed with known 2-methylbutyric acid. This finding is of importance, since the additional possibility arises that 2-methylbutyryl CoA might also serve to donate CoA to succinate in the Ascaris mitochondrion. Phosphorylation coupled to the decarboxylation of succinate in the Ascaris mitochondrial soluble fraction. All of the enzymatic activities required for reactions [l] through [4] are present in Ascaris mitochondria. It is necessary, therefore, to demonstrate that the esterification of inorganic phosphate to form organic phosphate was coupled to the decarboxylation of succinate. Indeed, this was demonstrable. .A dialyzed, soluble fraction obtained from Ascaris mitochondria catalyzed the incorporation of a low level of inorganic 32P into organic phosphate when incubated with either succinate, propionyl CoA, or succinate plus ATP (Table IV). Of greatest significance was the finding that the addition of propionyl CoA to succinate resulted in a greater than &fold increase in 32P incorporation. This increase was noted in seven different experiments (range = 3.2to 8.2-fold increase; average = 4.8-fold). Such an increase would be predicted on the basis of reactions [l] through [4]. That propionyl CoA is giving rise to succinyl CoA in these experiments was further indicated by the findings that succinyl CoA is the preferred substrate for 32P incorporation, and the addition of CoA + ATP to succinate resulted in an approximate 3.5-fold stimulation. Most important, avidin inhibited the propionyl CoA-stimulated incorporation of 32P approximately 85% indicating the participation of a biotin enzyme, such as propionyl CoA carboxylase, in this sequence of reactions. DISCUSSION

Ascaris lumbricoides var. suum muscle mitochondria have been shown to contain propionyl CoA carboxylase, methylmalonyl CoA mutase, and acyl CoA transferase activities. These activities are required for the postulated pathway of formation of

TABLE

IV

PHOSPHORYLATION COUPLED TO THE DECARBOXYLATION OF SUCCINATE IN THE Ascaris DIALYZED MITOCHONDRIAL SYSTEM AND THE EFFECT OF PROPIONYL COA”

Additions Succinate Succinate + propionyl CoA Propionyl CoA Succinate + CoA + ATP Succinate + ATP Succinyl CoA Succinate + propionyl CoA + avidin

3pP Incorporation into organic phosphate (nmol/20 minimg protein) 3.6 29.5 3.6 12.5 4.5 84.8 7.4

’ In addition to 1.1 mg of protein, each vessel contained in a total volume of 1.0 ml the following components, in pmol: Tris-HCl, pH = 7.4,40; glucose, 10; ADP, 2; MgCI,, 10; coenzyme B,*, 2; reduced glutathione, 5; potassium chloride, 20; inorganic, 32P, 12 (specific radioactivity = 187,000 dpm/pmol). Where indicated, vessels contained the following: sodium succinate, 10; propionyl CoA, 2; succinyl CoA, 2; coenzyme A, 2; ATP, 4; avidin, 0.05 mg/ml. 0.28 U of yeast hexokinase was added to all vessels which were incubated at 30°C for 20 min under an atmosphere of N,. Reactions were stopped by the addition of 0.11 ml of 40% trichloroacetic acid. Phosphorylation was then determined as described in the text.

propionate, a major product of Ascaris fermentation, from succinate. Earlier findings (11) employing Ascaris muscle strips incubated with 14C-labeled succinate, propionate, or CO*, respectively, demonstrated the reversible interconversion of succinate and propionate. In the disrupted mitochondrial preparations employed in these studies, this conversion of succinate to propionate is coupled to a substrate level phosphorylation which could be of vital importance to the economy of the helminth. The possibility exists that succinyl CoA formed in these experiments from succinate + propionyl CoA may give rise to ATP via a succinyl-thiokinase reaction. However, this does not appear likely in view of the specific inhibitory effects of avidin on both succinate decarboxylation as well as 32P esterification and the formation of

PHOSPHORYLATIONSUCCINATE

DECARBOXYLATION

propionate in these reactions. Unfortunately, the lack of permeability of the mitochondrial membranes to CoA derivatives precluded demonstrating phosphorylation in intact mitochondria. Ascaris has served as a model system, since it was the first organism shown to catalyze an electron transport-associated ATP generation which was coupled to succinate formation via the fumarate reductase reaction (11, 13, 19-22). More recently, additional helminths, other invertebrates, vertebrates, bacteria, and mammals have been shown or suggested to employ a similar energy-generating fumarate reductase pathway (2, 5, 22-24). The finding of a second site for ATP generation in these anaerobically functioning mitochondria is of considerable interest from numerous points of view. Many anthelmintic agents have been shown to inhibit the fumarate reductase system and the associated phosphorylation (25-28). Inhibitors of the propionate-forming system may prove to exhibit similar chemotherapeutic effects on infections with propionate-forming helminths. This possibility is particularly intriguing, since the methylmalonyl CoA mutase reaction requires vitamin Blz coenzyme, and wherever determined this vitamin has been found in physiologically significant amounts only in those helminths which form propionate (6,29). Those species which do not contain vitamin B,, form primarily succinate, lactate, or products unrelated to propionate. ACKNOWLEDGMENT The authors Gail A. Dunbar

wish to express their gratitude to for her excellent technical assistance. REFERENCES

1. KAZIRO, Y., AND OCHOA, S (1961) J. Biol. Chem. 236,3131-3136. 2. MACY, J. M., LJUNGDAHL, L. G., AND GOTTSCHALK, G. (1978) J. Bacterial. 134, 84-91. 3. DELWICHE, E. A., PHARES, E. F., AND CARSON, S. F. (1956) J. Bacterial. 71, 598-603. 4. VON BRAND, T. (1973) Biochemistry of Parasites, 2nd ed., p. 120, Academic Press, New York. 5. GADE, G., WILPS, H., KHUYTMANS, J. H. F. IN., AND DEZWAAN, A. (1975). J. Comp. Physiol. 104, 79-85.

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