Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria

Comp. Biochem. Physiol. Vol. 83B, No. 3, pp. 523-527, 1986 Printed in Great Britain

0305-0491/86 $3.00+0.00 ~ 1986 Pergamon Press Ltd

PURIFICATION A N D PROPERTIES OF A N ACYL CoA TRANSFERASE FROM A S C A R I S S U U M MUSCLE MITOCHONDRIA* GERALD L. McLAUGHLIN,% HOWARD J. SAZ~ and BECKY S. DEBRUYN Department of Biological Sciences University of Notre Dame, Notre Dame, IN 46556 USA (Tel: 219-239-6552) Received 24 July 1985) Abstract--l. An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. 2. From SDS PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent tool. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 + 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. 3. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. 4. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.

INTRODUCTION

(3) methylmalonyl C o A + E

Ascaris lumbricoides var. suum ferments carbohydrates to succinate plus a number of volatile acids, which include acetate, propionate, n-valerate, 2-methylbutyrate (2-MB) and 2-methylvalerate (2-MV). Succinate, 2-MB and 2 - M W constitute the quantitatively major fermentation products. In the formation of succinate by Ascaris and numerous other organisms, it is generally accepted that electrontransport associated generation of A T P is coupled to the anaerobic reduction of fumarate (Saz, 1971, 1981). More recently, Saz and Pietrzak (1980) and Pietrzak and Saz (1981) reported evidence supporting the concept that the decarboxylation of succinate to proprionate is coupled to a substrate level production of A T P in muscle mitochondria not only from the nematode, Asearis but also from the cestode Spirometra mansonoides and the trematode, Fasciola hepatica, according to the following sequence of reactions:

prop. CoA Carboxylase

" " C O / ' ~ E + propionyl C o A

(4) " C O " ~ E + A D P + P~ prop. CoA carboxylase

CO2 + A T P + E Sum (1)-(4): succinate + A D P + Pi ~ p r o p i o n a t e + CO2 + ATP. Each of these reactions was shown to be present in high activity in crude disrupted mitochondria prepared from all three of the parasites. Reaction (1), an acyl C o A transferase, is required for the activation of metabolically formed succinate via the acyl C o A derivative. Therefore, attempts were made to purify and examine the acyl C o A transferase from Asearis mitochondria which might be responsible for the activation of succinate. MATERIALS

(1) propionyl C o A + succinate transferase

" succinyl C o A + propionate (2) Succinyl C o A mutase + coenzyme BI2

• methylmalonyl C o A

*Supported in part by Grants AI-09483 and A1-07030 from the National Institutes of Health, United States Department of Health, Education and Welfare. tPresent address: Malaria Branch C-22, Center for Disease Control, 1600 Clifton Rd. NE, Atlanta, GA 30333, USA. :~To whom requests for reprints should be addressed. 523

AND

METHODS

A rapid spectrophotometric assay for acyl CoA transferase was developed by employing acetate as the CoA acceptor and coupling the acetyl CoA formed with oxaloacetate plus citrate synthase. This resulted in the formation of citrate and free coenzyme A. The free coenzyme A was then determined with the dye DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] according to Studier (1973). Each vessel contained (/lmol): Tris-Cl buffer, (pH = 8.0) 72; sodium acetate, 14.4 propionyl CoA, 0.14; oxaloacetate, 0.72; DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] 0.36. In addition, vessels contained 0.5 units of citrate synthase (Boehringer, Mannheim, FRG) and acyl CoA transferase activity sufficient to result in optical density changes between 0.02 and 0.17 units/min. The final volume was 0.8 ml. Changes in optical density were followed every 30 sec for 2 min at 412/~m. In the absence of an acceptor the

524

GERALD L. MCLAUGHLINet al.

acyl CoA esters were stable, indicating the absence of interfering deaeylase activity. In addition, isolation of the CoA derivatives by HPLC agreed both qualitatively and quantitatively with the spectrophotometric assay. Both the spectrophotometric and HPLC assay were shown to be linear for the time period and enzyme concentrations employed. A more tedious assay had to be employed when acids other than acetate acted as CoA acceptors. In these instances the CoA esters were isolated and quantitated by means of HPLC (Saz and Pietrzak, 1980; Pietrzak and Saz, 1981). Incubations were performed routinely at 30'C in closed 1.5 ml plastic centrifuge tubes. In a final volume of 0.4ml, each vessel contained (#mol): MES buffer (pH = 6.5), 40; magnesium chloride, 2; acyl CoA donor, 0.12; neutralized acceptor, 80; sufficient acyl CoA transferase to only partially complete the reaction in the 15-min incubation period. Incubations were terminated by the addition of perchloric acid to a final concentration of 2%. After neutralization with KOH, the precipitated protein and potassium perchlorate were removed by centrifugation and the more polar compounds were separated from the supernatants by passage through Ct8 Sep-Pak cartridges (Waters, Inc). After methanol and water washes of the cartridges, the incubation supernatants were added to the column. The columns were then water washed with 2.0 ml and the acyl CoA esters were eluted from the Sep-Pak cartridges in I ml of 50% methanol. Aliquots of the Sep-Pak eluates were subjected to paired ion HPLC. The CoA esters were eluted with 45% methanol which contained P I C A (Waters Inc.) at a flow rate of 1.0 ml/min. Acyl CoA ester peaks were determined using a spectrophotometric detector at 254 nm and quantitated by means of triangulation and comparison with the corresponding known acyl CoA. The following relationship was employed: A - A+B

x 0.12 ~mol =/~mol of A formed,

where A = peak area of newly formed CoA ester, B = p e a k area of initial CoA ester and 0.12 #mol represent the initial quantity of CoA ester added to the incubation. Known CoA ester mixtures retained identical relative peak areas before and after Sep-Pak treatment. The molecular weight of Ascaris acyl CoA transferase was estimated by elution patterns from 1.5 x 60 cm columns of Sephadex G-100, Sephadex G-150, Sephacryl S-200 (Pharmacia) and Ultrogel AcA 34 (LKB). Gels were equilibrated with phosphate buffer (0.02 M, pH = 7.2) which contained 0.05 M NaC1 and 1 mM EDTA. Between 1 and 10 units of acyl CoA transferase were applied to the columns in 0.05-2.0 ml of buffer, and 0.05 ml aliquots were collected and analyzed for proteins and activity. Reference proteins and their (molecular weights) included ferritin (440,000), catalase (232,000), aldolase (158,000), bovine serum albumin (BSA) (67,000), ovalbumin (43,000) and cytochrome c (12,400). Aliquots of protein for sodium dodecyl sulfate polyaerylamide gel electrophoresis (SDS-PAGE) were stored at - 6 0 ° C until the day of electrophoresis. Immediately after thawing, samples were heated for 2 min at 98°C with the sample buffer which contained the final concentration indicated: 2% SDS (w/v), 2% mercaptoethanol (v/v), 10% glycerol (v/v), 5 mM EDTA, 0.0675 M Tris-HCl (pH = 6.8) and 0.001% bromophenol blue. Slab gel apparati were modified after Studier (1973). Gels were stained with either Coomassie Blue, or silver (Merril et al., 1981). Protein standards employed with SDS-PAGE included, myosin (200,000), /~-galactosidase (117,000), phosphorylase (94,000), BSA (67,000), catalase (58,000), ovalbumin

(43,000), aldolase (39,000), chymotrypsin (22,000) and ribonuclease (13,500). The isoelectric point (pl) of Ascaris acyl CoA transferase was determined employing a chromatofocusing column (Pharmacia). Purified acyl CoA transferase (2.1 units) was applied in 1.5 ml of 0.02 M potassium phosphate buffer, pH - 7.4 and the column was eluted with 200 ml of degassed Polybuffer 74 (initial pH = 4.0 with HC1). Enzyme assays and pH measurements were performed for each fraction. The pI also was estimated by slab gel isoelectric focusing. DTNB was purchased from Aldrich Chemical Co., Milwaukee, WI. Oxaloacetate, citrate synthase, coenzyme A and all of its acyl CoA derivatives other than those of the branched chain volatile acids were obtained from Pharmacia-PL, Piscataway, NJ. The branched chain acyl CoA derivatives were synthesized according to Kawaguchi et al. (1981). RESULTS Puril~'cation a n d characterization chondrial acyl CoA transferase

q [ Ascaris m i t o -

M i t o c h o n d r i a from 4 0 g of Ascaris lumbricoides var. suum muscle were o b t a i n e d essentially according to Saz a n d Lescure (1969). The m i t o c h o n d r i a l medium c o n t a i n e d 0.24 M sucrose, 5 m M E D T A a n d 0.15% (w/v) bovine serum a l b u m i n titrated to pH = 7.4. The washed m i t o c h o n d r i a were suspended in 8 ml of 0.02 M M E S buffer (pH = 7.2) which contained 1 m M E D T A and frozen until required. T h a w e d m i t o c h o n d r i a l suspensions were disrupted further by sonication employing a B r a n s o n Sonifier at a 60 W power setting with the micro-tip. The suspension was subjected to five successive 15-sec bursts with 15-sec cooling periods. Insoluble comp o n e n t s were removed by centrifugation at 129,000 g for 60rain. The s u p e r n a t a n t was collected and employed as the starting material for further purification. A m m o n i u m sulfate fractionation was accomplished employing a saturated solution at 4 C which was neutralized with NH4 OH. The pellet precipitating between 56 and 77% saturated a m m o n i u m sulfate was b r o u g h t to 2.0 ml with 0.02 M potassium p h o s p h a t e buffer (pH = 7.1) which contained 1 m M E D T A . The fraction was desalted with the aid of a P h a r m a c i a PD-10 Sephadex G.25 column which h a d been equilibrated with the same buffer. The eluate was applied to a 1.5 x 5 cm D E A E - 5 2 column (Whatm a n preswollen DE-52) which had been equilibrated with the above p h o s p h a t e E D T A buffer. The elution proceeded stepwise with 0.02, 0.04 and finally 0.15 M potassium p h o s p h a t e buffer containing 1 m M E D T A , pH = 7.1. The 0.15 M acyl C o A transferase eluting buffer (approx. 4.0 ml) was divided into two equal aliquots, each of which was desalted on separate P D 10 Sephadex G-25 columns equilibrated with 0.01 m M E D T A in 0.002 M potassium p h o s p h a t e buffer, pH = 7.1. Each desalted fraction was applied, in turn, to a 1.0 x 4.0 cm Cj0 agarose h y d r o p h o b i c column (Miles-Yeda Ltd., Elkhart, IN) which had been equilibrated with the above 0.01 m M E D T A + 0.002 M p h o s p h a t e buffer, pH = 7.1. After washing with several c o l u m n volumes of the equilibration buffer, the acyl C o A transferase was eluted with 0 . 0 6 M p h o s p h a t e (pH = 7 . 1 ) containing 0.01 m M E D T A . The molarity of the buffer required

Ascaris

acyl CoA transferase

525

Table 1. Purification of acyl CoA transferase from Ascaris mitochondria Total Specific Protein activity* activity* Recovery* Fraction (rag) (/~mol/min) /~mol/min/mg) (%) Mitochondrial supernatant 33 40 1.2 100 Ammonium sulfate 9.7 28 + 5 2.9 _+0.2 70 + 14 DEAE-52 4.8 22 _+4 4.6 _+0.8 55 _+9 Hydrophobic C~0 0.11 12_+2 109_+4 30_+5 *Where indicated. _+SE of the mean; N = 6. for elution of the enzyme tended to decrease with increasing age of the column. Employing this procedure a 90-fold purification was obtained over the initial mitochondrial supernatant with an approximate 30% yield of activity (Table 1). Purification also was followed by SDSP A G E (Fig. 1) which indicated that the fraction obtained from the C10 hydrophobic column was homogeneous, containing only the acyl C o A transferase protein. By comparison with standard proteins,

the SDS gel electrophoresis suggested an apparent molecular weight of the purified peptide of 50,000. Molecular exclusion chromatography on Sephadex G-100 (Fig. 2), Ultrogel A c A 34 and Sephadex G-150 indicated molecular weights of the intact acyl C o A transferase of 109,000, 100,000 and 101,000 respectively. The finding of the apparent mol. wt of 50,000 after S D S - P A G E would be in agreement with the enzyme being composed of two similar subunits each having a mol. wt of approx 50,000.

Fig. 1. SDS-PAGE of fractions during purification of Ascaris acyl CoA transferase. Tracks: 1, soluble mitochondrial fraction; 2, 5(~77% ammonium sulfate fraction; 3, eluate from DEAE-52 column; 4, eluate from hydrophobic C,0 column. Molecular weight standards were myosin,/~-galactosidase, phosphorylase, bovine serum albumin, ovalbumin, and chymotrypsin (top to bottom).

526

GERALD L. MCLAUGHLIN e t a / .

12.0 ~

~'~(

I)

I0.0

:~ 8.0

~__.xcoA rR~SFERASE

6.0

4.0

~41 J

4.5

5.~0

-..~5) 515

LOG MOLECULAR WEIGHT Fig. 2. Sephadex G - I 0 0 c h r o m a t o g r a p h y of purified Ascaris m i t o c h o n d r i a l acyl C o A transferase and s t a n d a r d proteins. (1) c y t o c h r o m e c; (2) o v a l b u m i n ; (3) bovine serum a l b u m i n ; (4) aldolase; (5) catalase. C o A transferase is s h o w n at the arrow.

As determined by column chromatofocusing, the isoelectric point (pl) of the purified acyl CoA transferase was 5.6 +_ 0.1 pH unit. Consistently, for both crude and purified enzyme sources, transferasc activity was found only in this pH region. Similar p| values were determined using flat bed isoelectric focusing. In these experiments, activities were determined spectrophotometrically employing DTNB.

CoA donor and acceptor specOqcities q / the Ascaris acyl CoA transferase The purified Ascaris mitochondrial acyl CoA transferase was relatively specific for the normal low

molecular weight straight-chain monocarboxylic acids as both donors and acceptors (Table 2). Acetyl-, propionyl-, n-butyryl-, n-valeryl- and hexanoyl CoA esters are highly active. On the other hand, the branched-chain acyl CoA derivatives as well as the unsaturated compounds (tiglyl- and crotonyl CoA) were inactive, or possessed minimal activities as CoA donors. Similarly, the long chain acyl CoA esters, such as palmitoyt-, stearoyl- and archidonyl CoA were inactive. Acetoacetyl CoA exhibited a low activity of questionable significance. Acetate and propionate were far superior to other acids tested as CoA acceptors. Although some activity was exhibited with some other acceptors, the relative reaction rates were much lower than displayed by acetate and propionate. Most interesting, neither succinyl CoA nor succinate exhibited appreciable activities as CoA donor or acceptor, respectively.

DISCUSSION

An acyl CoA transferase has been purified to electrophoretic homogeneity from Ascaris suum mitochondria. The enzyme appears to be composed of two similar or identical subunits with mol. wts of approx. 50,000 and a holoenzyme of approx. 100,000. Substrate specificity studies indicated that the transferase was specific for both the lower straight-chain acylmoiety as CoA donors and the straight chain acceptors. The branched-chain acyl CoA derivatives

Table 2. Donor and acceptor specificities of purified Ascaris mitochondrial acyl CoA transferase Donor

Acceptor

Activity (/~mol/min/mg protein)

Acetyl CoA

Propionate Succinate

194 4

Propionyl CoA

Acetate Succinate Butyrate n-Valerate 2-Methylbutyrate 2-Methylvalerate

n-Butyryl CoA

Propionate Succinate

102 0.3

n-Valeryl CoA

Propionate Succinate

138 0.3

2-Methylbutyryl CoA

Acetate Propionate Succinate

14 9 2

Tiglyl CoA

Acetate Propionate Succinate

0 0 0

Succinyl CoA

Acetate Propionate 2-Methylbutyrate 2-Methylvalerate

0 0.2 0 0

n-Hexanoyl CoA

Acetate

245

iso-Butyryl CoA

Acetate Propionate

240 26

iso-Valeryl CoA Acetoacetyl CoA Crotonyl CoA /~-Methylcrotonyl CoA Palmitoyl CoA Stearoyl CoA Arachidonyl CoA

Acetate Propionate Propionate Succinate Acetate Acetate Acetate

33 5 2 0 0 0 0

99 6 18 10 11 1I

Ascaris acyl CoA transferase

exhibited either no or low activities, indicating that 2-methylbutyryl CoA and presumably 2-methylvaleryl CoA could not transfer the CoA moiety, in spite of the fact that 2-MB and 2-MV are major fermentation products. Most striking was the finding that this acyl CoA transferase was essentially not capable of catalyzing the transfer of CoA to succinate. Conversely, succinyl CoA was a poor donor. Therefore, this acyl CoA transferase cannot serve physiologically to activate succinate which is formed from fumarate as catalyzed by the fumarate reductase reaction in the normal metabolism of Ascaris muscle. Physiologically, it must serve in reactions other than the ATP generating decarboxylation of succinate to propionate as illustrated above. This transferase is quite active in the transfer of CoA from acetyl CoA to propionyl CoA. Acetyl CoA is formed in Ascaris muscle mitochondria by the pyruvate dehydrogenase reaction (Komuniecki et al., 1979), and serves as a precursor for one of the major fermentation products, 2-methylbutyrate (Saz and Weft, 1960). In addition, free acetate is a fermentation product. The possibility arises that this transferase serves to insure a source of propionyl CoA for the hypothesized series of reactions. In this sequence of reactions, only catalytic quantities of propionyl CoA are required, since it is regenerated upon the decarboxylation of succinate. However, propionyl CoA also serves as a precursor for both 2-methylbutyrate and 2-methylvalerate (Saz and Well, 1960, 1962). Free propionate also is elaborated as a carbohydrate fermentation product. Although crude preparations of disrupted Ascaris muscle mitochondria have been shown to catalyze a transfer of CoA from propionyl CoA to succinate, the acyl CoA transferase purified in these studies does not catalyze this reaction to any appreciable extent. It would appear, therefore, that one or more other transferases are present in the mitochondrion which are capable of catalyzing the transfer of CoA to succinate. The presence of such an enzyme activity has been confirmed and separated from the acyl CoA

C.B.P. 8 3 / 3 B ~

527

transferase reported in these investigations (Saz and deBruyn, unpublished observations). REFERENCES

Bradford M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Analyt. Biochem. 72, 248-256. Kawaguchi A., Yoshimura T. and Okuda S. (1981) A new method for the preparation of acyl-CoA thioesters. J. Biochem. 89 337-339. Komuniecki R., Komunieki P. R. and Saz H. J. (1979) Purification and properties of the Ascaris.pyruvate dehydrogenase complex. Biochim, biophys. Acta 571, 1-11. Merril C. R., Switzer R. C. and Van Keuren, M. L. (1981) Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc. natn. Acad. Sci. U.S.A. 76, 43354339. Pietrzak S. M. and Saz, H. J. (1981) Succinate decarboxylation to propionate and the associated phosphorylation in Fasciola hepatica and Spirometra mansonoides. Molec. biochem. Parasitol. 3, 61-70. Saz H. J, (1971) Anaerobic phosphorylation in Ascaris mitochondria and the effects of anthelmintics. Comp. Biochem. Physiol. 39B, 627-637. Saz H. J. (I 981) Energy metabolisms of parasitic helminths: adaptations to parasitism, A. Rev. Physiol. 43, 323-341. Saz H. J. and Lescure, O. L. (1969) The functions of phosphoenolpyruvate carboxykinase and malic enzyme in the anaerobic formation of succinate by Ascaris lumbricoides. Comp. Biochem. Physiol. 30, 49-60. Saz H. J, and Pietrzak S. M. (1980) Phosphorylation associated with succinate decarboxylation in Ascaris mitochondria. Archs Biochem. Biophys. 202, 388-395. Saz H. J. and Well A. (1960) The mechanism of the formation of ~-methylbutyrate from carbohydrate by Ascaris lumbricoides muscle. J. biol. Chem. 235, 914-918. Saz H. J. and Well A. (1962) Pathway of formation of ct-methylvalerate by Ascaris lumbricoides. J. biol. Chem. 237, 2053-2056. Srere P. A. (1969) Citrate synthase. In Methods in Enzymology, Vol. XIII (Edited by Lowenstein J. M.), pp. 3-11. Academic Press, New York. Studier F. W. (1973) Analysis of early T7 RNA's and proteins. J. molec. Biol. 79, 237-248.