Identification of a novel dihydrolipoyl dehydrogenase-binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode, Ascaris suum.

A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which lacks an amino-terminal lipoyl domain, p45, has been identified in the pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited significant similarity with internal E3-binding domains of dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution of a pyruvate dehydrogenase-depleted adult A. suum PDC in guanidine hydrochloride resulted in two E3-depleted E2 core preparations which were either enriched or substantially depleted of p45. Following reconstitution, the p45-enriched E2 core exhibited enhanced E3 binding, whereas, the p45-depleted E2 core exhibited dramatically reduced E3 binding. Reconstitution of either the bovine kidney or A. suum PDCs with the A. suum E3 suggested that the ascarid E3 was more sensitive to NADH inhibition when bound to the bovine kidney core. The expression of p45 was developmentally regulated and p45 was most abundant in anaerobic muscle. In contrast, E3s isolated from anaerobic muscle or aerobic second-stage larvae were identical. These results suggest that during the transition to anaerobic metabolism, E3 remains unchanged, but it appears that a novel E3BP, p45, is expressed which may help to maintain the activity of the PDC in the face of the elevated intramitochondrial NADH/NAD ratios associated with anaerobiosis.

The pyruvate dehydrogenase complex (PDC) 1 plays a key role in the unique mitochondrial metabolism of the parasitic nematode, Ascaris suum (1,2). During larval development, A. suum exhibits a marked aerobic-anaerobic transition in energy metabolism. Early larval stages are aerobic, and the PDC functions to supply acetyl-CoA for tricarboxylic acid cycle oxidation (3). In contrast, energy metabolism in adult ascarid muscle is anaerobic. Adult muscle mitochondria lack both a functional tricarboxylic acid cycle and cytochrome oxidase activity and use unsaturated organic acids, such as fumarate and 2-methyl branched-chain enoyl-CoAs, instead of oxygen, as terminal electron acceptors (4 -6). The PDC is overexpressed in adult muscle and is designed to function under the elevated NADH/ NAD ϩ and acyl-CoA/CoA ratios present in the microaerobic environment of the host gut (7,8).
The subunit composition of the adult ascarid muscle PDC differs significantly from the PDCs isolated from other eukaryotic organisms (7,9,10). In both yeast and mammalian PDCs, incubation of the complex with [2-14 C]pyruvate in the absence of CoA results in the acetylation of two proteins, dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3)binding protein (BP) (11,12). E2 and E3BP are present in a ratio of about 5:1, and both contain similar multidomain structures consisting of an amino-terminal lipoyl domain, a subunit binding domain, and an inner domain (13)(14)(15). E2 preferentially binds pyruvate dehydrogenase (E1) and exhibits transacetylase activity, whereas E3BP contains a high-affinity binding site for E3 (16). In contrast, incubation of the ascarid complex under similar conditions results in the acetylation of only E2 and no protein corresponding to E3BP has been identified (10). In addition, the PDC from A. suum and other closely related nematodes contains subunits of 43 kDa (p43) and 45 kDa (p45) of unknown function, which do not appear to be present in PDCs from other organisms (17). Whether the E3binding site in the ascarid PDC resides in E2, as has been observed for PDCs from prokaryotes, or in one of these novel proteins has not been determined (18).
In the present study, we report that sequence near the amino terminus of p45 exhibits significant similarity to the putative E3-binding domains of E2 and E3BP and demonstrate that E3 binding to the E2 core is dependent on the presence of p45. In addition, we show that ascarid E3 bound to the bovine kidney core (containing E3BP) appears to be more sensitive to NADH inhibition than when it is bound to the ascarid core (containing p45). Therefore, it appears that during the switch from aerobic to anaerobic energy metabolism in ascarid development, E3 remains unchanged, but a novel anaerobic E3BP, p45, is expressed to maintain PDC activity in the face of elevated NADH/ NAD ϩ ratios.

Materials
A. suum were obtained from Routh Packing (Sandusky, OH). PDC (3.5 mol of NADH min Ϫ1 (mg of protein) Ϫ1 ) was isolated from frozen A. suum muscle strips as described previously (19). A. suum E3 was prepared as described previously (20). A. suum larval stages were prepared as described previously (21). Bovine kidney PDC, E2⅐E3BP core, and E1 were the generous gift of Dr. Thomas E. Roche (Kansas State University, Manhattan, KS). All chromatographic matrices were purchased from Pharmacia Biotech (Piscataway, NJ), and all electrophoresis reagents were from Bio-Rad. All other chemicals and reagents were of the highest grade available and purchased from Sigma.

Methods
Sequential Dissociation and Resolution of A. suum PDC-A. suum E1-depleted PDC and E1 were prepared as described previously (10) with the following modification: dissociation was performed in 50 mM MOPS (pH 7.4), 2 M NaCl, 1 mM MgCl 2 , 0.1 mM EDTA, 3 mM dithiothreitol, and 0.8 mg ml Ϫ1 Pluronic ® F68 (BASF Corp.). After incubation at room temperature for 90 min, the sample was applied to a Sephacryl S-400 column (2.6 ϫ 90 cm) equilibrated with the same buffer. The first peak contained E1-depleted PDC and was concentrated by centrifugation at 155,000 ϫ g for 3 h. The pellet was resuspended in 50 mM MOPS (pH 7.4), 1 mM MgCl 2 , 1 mM EDTA, 0.6 mg ml Ϫ1 Pluronic ® F68, and 3 mM dithiothreitol (Buffer A). E1-depleted PDC (0.5-0.8 mg) was incubated for 60 min at room temperature in 50 mM MOPS (pH 7.4), 1 M GdnHCl (Pierce), 1 mg ml Ϫ1 Pluronic ® F68, 1 mM EDTA, and 3 mM dithiothreitol (final volume 250 l), followed immediately by fast protein liquid chromatography on a Superose 12 HR10/30 column equilibrated in the same buffer. Peak fractions were pooled. After removal of GdnHCl by dialysis, Peak 1 was centrifuged at 155,000 ϫ g for 4 h. The pellet (Peak 1, see Fig. 1) was insoluble using a variety of solubilization techniques including detergents, heparin, and NH 4 Cl. Therefore, following centrifugation the pellet was resuspended directly in Laemmli sample buffer for SDS-polyacrylamide gel electrophoresis (PAGE). Peaks 2 and 3 were desalted and concentrated in a Centricon ® 30 (Amicon Inc.) with several changes of Buffer A. Protein was determined according to Bradford using bovine serum albumin as a standard (22).
Reconstitution and Binding of Resolved Components following Guanidine Hydrochloride Dissociation-After resolution and the removal of 1 M GdnHCl, Peak 2 (100 g) was incubated with excess A. suum E1 (200 g) and E3 (100 g) for 30 min at room temperature in Buffer A (final volume of 2.5 ml). PDC activity of the reconstituted sample was measured spectrophotometrically as described previously (7). The reconstituted sample was layered over 4 ml of 20% (w/v) sucrose in Buffer A. After centrifugation at 155,000 ϫ g for 5 h, the pellet was resuspended in Buffer A and the supernatant was concentrated by Centricon ® 30 with several changes of Buffer A. Since Peak 1 was insoluble after removal of GdnHCl, Peak 1 (approximately 150 g) was dialyzed against 50 mM MOPS (pH 7.4), 1 mM EDTA, and 3 mM dithiothreitol (Buffer B) containing 0.5 M GdnHCl. After 2 h, E3 (100 g) was added and the sample was dialyzed against Buffer B containing 0.1 M GdnHCl for an additional 3 h. After a final dialysis against Buffer B containing 0.02 M GdnHCl for 6 h, the sample was layered over 4.0 ml of 20% (w/v) sucrose in Buffer A and centrifuged at 155,000 ϫ g for 5 h. This pellet was resuspended directly in Laemmli sample buffer.
V8 Protease Treatment-E1-depleted PDC (500 g) was incubated in 15 mM Tris-HCl (pH 7.4), 3 mM MgCl 2 , 1 mM dithiothreitol, and 50 g of V8 protease (final volume of 0.5 ml). After incubation at room temperature for 60 min, the reaction was stopped with the addition of 3,4-dichloroisocoumarin (final concentration of 0.2 mM), and 400 l of the stopped reaction was layered over 2 ml of 15% (w/v) sucrose in 50 mM MOPS (pH 7.4), 1 mM dithiothreitol, and 0.1 mM EDTA. After centrifugation at 155,000 ϫ g for 3 h, the pellet was resuspended in 50 mM MOPS (pH 7.4), 1 mM dithiothreitol, and 0.1 mM EDTA (Buffer C) and the supernatant was concentrated in a Centricon ® 30 with several changes of Buffer C.
NADH Sensitivity of Native and Hybrid PDCs-A. suum PDC activity was measured spectrophotometrically as described previously (7). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.4), 1 mM MgCl 2 , 0.2 mM NAD ϩ , 2 mM dithiothreitol, 0.1 mM thiamine pyrophosphate, 0.1 mM CoA, and 4 mM pyruvate in a final volume of 1 ml. Bovine kidney PDC activity was measured spectrophotometrically as described previously (23). The reaction mixture contained 50 mM potassium phosphate (pH 8.0), 1 mM MgCl 2 , 2.5 mM NAD ϩ , 2.6 mM dithiothreitol, 0.2 mM thiamine pyrophosphate, 0.13 mM CoA, and 2 mM pyruvate in a final volume of 1 ml. A. suum E3 was prepared as described previously (20). The bovine kidney-A. suum hybrid complexes were prepared as follows: bovine kidney E2⅐E3BP (2 g) and bovine kidney E1 (4 g) were preincubated in 50 mM potassium phosphate (pH 8.0), 1 mM MgCl 2 , and 3 mM dithiothreitol at room temperature for 5 min. A. suum E3 (1 g) was then added and the preparation was incubated at room temperature for an additional 15 min. PDC activity of the hybrid complex was measured using the conditions described for the bovine kidney PDC (23). Sensitivity of the different complexes to NADH was determined using various concentrations of NAD ϩ and NADH (constant nicotinamide pool of 200 M) in the PDC activity assay, or a fixed NAD ϩ concentration (either 10 or 20 M) with various concentrations of NADH in the PDC activity assay.
Preparation of A. suum Larval Homogenates-A. suum unembryonated "eggs" (UE), first-stage (L1), second-stage (L2), or third-stage larvae (L3) were resuspended (1 g of larvae/4 ml of buffer) in 20 mM MOPS (pH 7.2), 2 mM EDTA, 2 mM EGTA, and a protease inhibitor mixture consisting of: 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2.1 M leupeptin, 3 M aprotinin, 1 M soybean trypsin inhibitor, 66 M antipain, 33 M chymostatin, 152 M ⑀-amino caproic acid, and 29 M pepstatin A. Homogenates were prepared by three passes through a French pressure cell at 20,000 p.s.i. Triton X-100 then was added to a final concentration of 1% (w/v). After 30 min on ice, samples were centrifuged for 30 min at 10,000 ϫ g and the supernatants frozen in liquid nitrogen. Samples were concentrated by ultrafiltration and centrifuged for 15 min at 9,000 ϫ g to remove insoluble material.
Purification of E3 from Second-stage Larvae-All procedures were performed at 4°C unless otherwise stated. L2s (50 ml) were washed with 20 mM potassium phosphate (pH 7.4) and resuspended in 20 mM potassium phosphate (pH 7.4), 2 mM EDTA, and protease inhibitor mixture, and passed through a French pressure cell at 20,000 p.s.i. three times. The homogenate was centrifuged at 1,000 ϫ g for 10 min, and the resulting supernatant was then centrifuged at 10,000 ϫ g for 30 min. The pellet was discarded and the supernatant was centrifuged for 3 h at 155,000 ϫ g. The resulting pellet was resuspended in 20 mM potassium phosphate (pH 7.4), 1 mM EDTA, 1 mM EGTA, and protease inhibitor mixture, incubated in a 70°C water bath for 10 min, and immediately cooled on ice for 15 min. Following an initial clarification at 9,600 ϫ g for 15 min, the supernatant was centrifuged again for 3 h at 155,000 ϫ g. The pellet was discarded and the supernatant was concentrated using Centricon ® 10 with the buffer exchanged to 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, and 1 mM EGTA (Buffer D). The concentrated sample was applied to a DEAE-Sepharose column (1 ϫ 7 cm) equilibrated with Buffer D. After washing with 15 ml of Buffer D, activity was eluted with a step-gradient comprised of Buffer D containing 75, 100, 150, and 200 mM NaCl, respectively. Activity eluted with 100 and 150 mM NaCl. Fractions with the highest specific activity were combined and applied directly to a hydroxylapatite column equilibrated with Buffer D. Activity eluted with 300 and 400 mM potassium phosphate (pH 7.2) was desalted and concentrated to 1 mg ml Ϫ1 and stored at Ϫ70°C. The purified E3 migrated as a single band with an apparent molecular mass of 55 kDa during SDS-PAGE.
Gel Electrophoresis and Immunoblotting-SDS-PAGE was conducted according to Laemmli (24) and proteins were visualized with Coomassie Blue R-250 staining.
For immunoblotting, samples were transferred to nitrocellulose (Schleicher and Schuell) overnight at 40 V in 25 mM Tris-HCl, 192 mM glycine, and 20% (v/v) methanol. To detect bands of interest, each specific antiserum was used in conjunction with the Promega kit, Protoblot ® Western blot AP system (Promega Corp.), using goat anti-rabbit IgG secondary antibody conjugated to alkaline phosphatase. The procedure was performed as described by the manufacturer. Affinity purified antisera against p45, E1␣, and E1␤ were prepared as described previously (10,25,26). E2 antiserum was affinity-purified in a manner previously described (25,27).
Determination of Amino-terminal Sequence-Samples were separated by SDS-PAGE on a 12% gel, and transferred to Problott TM membranes (Applied Biosystems) in 100 mM CAPS (pH 11), and 10% (v/v) methanol. After transfer for 90 min, the membranes were stained with Coomassie Blue R-250 as described by the manufacturer. Individual p45 bands were excised and stored at Ϫ20°C. Sequencing was performed by Andrew Brauer of ARIAD Pharmaceuticals Inc. (Cambridge, MA), and William Burkhart of Glaxo Wellcome Inc. (Durham, NC).

RESULTS
Identification of a Putative E3-binding Domain in p45-As described previously, incubation of the adult A. suum PDC with [2-14 C]pyruvate, followed by SDS-PAGE and autoradiography yields only a single acetylated subunit, suggesting that the lipoyl-containing E3BP observed in other eukaryotic PDCs was not present in the adult ascarid complex (10). However, analysis of one novel subunit of the ascarid complex, p45, has indicated that its amino-terminal sequence is similar to that of the internal E3-binding domains of E2, E3BP, and succinyltransferase from a variety of other organisms (Table I). While the sequence identity is limited, the conserved hydrophobic residues predicted to be involved in intrahelical contacts within the hydrophobic domain and the charged residues predicted to be involved in E3 binding are predominantly conserved (28). In addition, Pro at position 14 in the A. suum sequence also is conserved except in the E3BP sequence from Saccharomyces cerevisiae. However, the yeast E3BP sequence does contain a Pro upstream at position 142. It has previously been suggested that this Pro represents the amino-terminal boundary of the binding domain (28). In contrast, the results of the present study indicate that the p45 of the adult A. suum muscle PDC is an E3-binding protein with a novel domain structure.
Dissociation of E3 from the A. suum E2 Core and the Generation of two Distinct E2 Core Populations-Techniques which dissociate both E1 and E3 from the mammalian PDC result in the dissociation of only E1 from the ascarid complex (10). To generate an E2 core free of E1 and E3, E1-depleted PDC prepared by treatment with 2 M NaCl was incubated with 1 M GdnHCl followed by fast protein liquid chromatography on Superose 12. This procedure separated the E1-depleted PDC into three fractions and generated two E2 core populations (Fig. 1). Peak 1, which eluted with the void volume, contained E2 and was enriched in p43 and p45. After the removal of GdnHCl, this fraction was insoluble. In contrast, Peak 2 contained E2 and trace amounts of both E3 and p45, and remained soluble after the removal of GdnHCl. Peak 3 only contained E3. The selective removal of E1 and E3 by different reagents suggests that E1 and E3 have specific, but different, interactions with the E2 core. The GdnHCl concentration used in this study was critical. For example, treatment of the E1-depleted PDC with 0.7 M, instead of 1.0 M GdnHCl, released only E3 and had no observable effect on the ratio of E2 to p45 in the core (data not shown).
Removal of p45 from the Core Decreases the Binding of E3, but Not E1-Incubation of the soluble, p45-depleted E2 core (Peak 2 from the separation in GdnHCl described above; Fig. 2, lane 6) with both E1 and E3 restored a portion of the PDC activity (about 20%, 1.1-1.5 mol min Ϫ1 (mg protein) Ϫ1 ). Sedimentation of the reconstituted complex revealed that E1 binding appeared to be unaffected (Fig. 2, lane 1), while E3 binding was significantly diminished (Fig. 2, lane 7) and almost all of the E3 remained in the supernatant fraction (Fig. 2, lane 8). In contrast, incubation of the p45-enriched E2 core (Peak 1 from the separation in GdnHCl described above; Fig. 2, lane 3) with excess E3, followed by dialysis to remove GdnHCl, revealed that E3 binding to the core was enhanced (Fig. 2, lane 4). These results suggest that p45 plays a role in E3 binding.
p45 Remains Intact and Associated with the Core After the Removal of the Lipoyl Domains of E2 by Selective Proteolysis-The E1-depleted PDC was incubated with V8 protease and pelleted at 155,000 ϫ g for 3 h (Fig. 3). E2 was sensitive to proteolysis, while p45 and E3 were resistant. Analysis of the proteolytic products by amino-terminal sequencing and immunoblotting with affinity purified polyclonal antisera against

TABLE I Comparison of amino-terminal sequence of p45 with conserved amino acids of the putative dihydrolipoyl dehydrogenase-binding domain
The underline is the proposed contact sites at the interface with E3 (through conserved charged side chains) (27) and the bold indicates the conserved hydrophobic residues in the core at interhelical contacts between helices (27  either E2 or p45 revealed that E2 was digested to a mixture of E2 I (VAPPARVGVAATMAGPVXXGGFIDIPVSENR), E2 IB (APPNYHKP), and two fragments of E2 L (PDLPEHKKIPL-PALSPTM), while the breakdown of p45 was minimal (data not shown). E2 I , E2 IB , and p45 sedimented with the pellet, while E2 L was found in the supernatant fraction. These results confirm the tight association of p45 with the E2 oligomer. In contrast, E3 was present in both the pellet and supernatant fractions after proteolysis. These results suggest that in addition to p45, E2 also may play a role in the binding of E3 to the core of the ascarid complex.
NADH Inhibition of Native and Hybrid Complexes-To determine if p45 plays a role in the decreased NADH sensitivity of the adult A. suum muscle PDC, the NADH sensitivities of the native bovine kidney and adult A. suum PDCs were compared with that of a hybrid complex constructed using the bovine kidney E2⅐E3BP core and E1, and the A. suum E3 (Fig.  4). The native A. suum PDC exhibited an apparent K m for NAD ϩ of 12.0 Ϯ 0.7 M (M Ϯ S.E., n ϭ 6) which was significantly lower than that exhibited by either the bovine kidney (59.4 Ϯ 1.5 M, M Ϯ S.E., n ϭ 4) or hybrid complex containing the ascarid E3 (54.2 Ϯ 1.6 M, M Ϯ S.E., n ϭ 4). As reported previously and confirmed in the present study, the adult A.
suum muscle PDC was less sensitive to NADH inhibition than the bovine kidney PDC, when assayed at either a fixed NAD ϩ or total nicotinamide nucleotide pool (Fig. 4). In fact, the A. suum PDC appears to be the most insensitive to NADH inhibition of all PDCs studied thus far (29). Most important, the hybrid complex constructed with bovine kidney E2⅐E3BP and E1, and ascarid E3 was more sensitive to NADH inhibition than the native A. suum PDC (Fig. 4). An NADH/NAD ϩ ratio of about 2 was required for 50% inhibition of the native A. suum PDC, while a ratio of about 1 was required for either the bovine kidney or hybrid complexes (Fig. 4). These results suggest that the binding of E3 to p45 may decrease the sensitivity of the complex to NADH inhibition. It should be noted that the assay conditions for the different PDCs were selected to maintain the activity of other components such as the rate-limiting E1 and maximize overall PDC activity. Therefore, the A. suum PDC was assayed at pH 7.4 and the bovine kidney and hybrid complexes at pH 8.0. Importantly, changing the pH of the assay buffer from 7.4 to 8.0 had only a negligible effect on the apparent K m for NAD ϩ and NADH inhibition for the native ascarid PDC, suggesting that the differences in E3 inhibition did not FIG. 2. Binding of A. suum peripheral components (E1 and E3) by two E2 core populations. A. suum p45-enriched E2 core (Peak 1) was reconstituted with excess A. suum E3, and p45-depleted A. suum E2 core (Peak 2) was reconstituted with excess A. suum E1 and E3 as described under "Experimental Procedures." Samples were separated by SDS-PAGE on 10% gels and stained with Coomassie Brilliant Blue. Lane 1, adult A. suum PDC (12 g); Lane 2, E1-depleted A. suum PDC (10 g); Lane 3, p45-enriched E2 core (Peak 1) (ϳ8 g); Lane 4, reconstituted Peak 1 pellet (ϳ12 g); Lane 5, reconstituted Peak 1 supernatant (4 g); Lane 6, p45-depleted A. suum core (Peak 2) (8 g); Lane 7, reconstituted Peak 2 pellet (10 g); and Lane 8, reconstituted Peak 2 supernatant (5 g).

FIG. 3.
Association of p45 with the core of the complex after selective proteolysis. E1-depleted A. suum PDC was treated with V8 protease and the proteolytic products were separated as described under "Experimental Procedures." Samples (9 g) were separated by SDS-PAGE on 12% gels and stained with Coomassie Brilliant Blue. The identity of E2 domain fragments was determined by immunoblotting and amino-terminal sequencing. Lane 1, E1-depleted PDC; Lane 2, E1-depleted PDC following proteolysis; Lane 3, pellet; Lane 4, supernatant.
FIG . 4. NADH sensitivity of A. suum, bovine kidney, and hybrid pyruvate dehydrogenase complexes. Hybrid complexes were constructed as described under "Experimental Procedures." A, PDC activity is expressed as a percentage of the activity measured in the absence of NADH using a fixed NAD ϩ concentration with varying NADH concentrations. B, PDC activity is expressed as a percentage of the activity measured in the absence of NADH using conditions of a fixed nicotinamide pool (200 M). Closed circles, A. suum PDC; squares, bovine kidney PDC; and open circles, bovine kidney E2⅐E3BP⅐E1/A. suum E3 hybrid complex. result from the different assay conditions.
Regulation of the PDC during Ascarid Larval Development-The role of the PDC during A. suum larval development changes dramatically and stage-specific, aerobic and anaerobic, isozymes of many mitochondrial enzymes have been identified previously (25,30,31). To examine the role of p45 during this aerobic/anaerobic transition, ascarid larval homogenates were immunoblotted with affinity purified polyclonal antisera against p45 and other subunits of the adult A. suum PDC (Fig.  5). All ascarid larval stages contained PDC, as evidenced by the specific staining with E2 antisera. However, as predicted, p45 appeared to be much less abundant or absent in the aerobic early larval stages (UE, L1, and L2). Previous work reported that p45 also is absent from the PDC of adult ovaries and testis (10).
Perhaps surprisingly, significant p45 staining was observed in homogenates of third-stage larvae (L3), even though they are aerobic and cyanide-sensitive. However, other key enzymes of anaerobic metabolism, such as the 2-methyl branched-chain enoyl-CoA reductase also have been identified in this transitional larval stage. Their expression may represent an adaptation to the switch to anaerobiosis that accompanies the impending molt to the fourth-stage (30). Taken together, these results suggest that p45 is found predominantly in anaerobic muscle. In addition, antisera against the adult muscle E1␣ and E1␤ recognize proteins of different mobilities in the aerobic larval stages. This confirms the presence of stage-specific E1␣ isozymes and further suggests that stage-specific forms of E1␤ also may be present.
To determine if E3s also were different in aerobic and anaerobic stages, E3 was purified to homogeneity from aerobic L2 (Table II). E3 from L2 behaved similarly to the E3 from adult muscle during purification and had identical kinetic properties and subunit molecular mass (55 kDa, data not shown). More important, amino-terminal sequence analysis of both E3s indicated that the first 53 amino acids were identical, suggesting that stage-specific isoforms of E3 were not present in the A. suum PDC (Table III). Therefore, it appears that during the switch from aerobic to anaerobic energy metabolism in ascarid development, E3 remains unchanged, but a novel anaerobic E3BP, p45, is expressed to maintain PDC activity in the face of elevated NADH/NAD ϩ ratios observed in adult body wall muscle. DISCUSSION A. suum undergoes a marked aerobic/anaerobic transition during its development and energy metabolism in adult muscle mitochondria is anaerobic and characterized by elevated NADH/NAD ϩ ratios (3). The results of the present study suggest that E3 is identical in both aerobic and anaerobic stages, but that the PDC from anaerobic mitochondria has a novel E3BP, p45. In contrast to E3BPs identified in either yeast or mammalian PDCs, p45 is not acetylated during incubation in [2-14 C]pyruvate and does not contain the highly conserved amino-terminal PS/ALSPTM sequence characteristic of lipoyl domains (10,32). In contrast, the amino terminus of p45 does contain sequence which exhibits significant similarity to E3binding domains found internally in E2s, E3BPs, and succinyltransferases isolated from a number of organisms. However, it should be noted that the E3-binding domain of p45 also exhibits significant differences from these putative E3-binding domains. Supporting the proposed role of p45 as an E3-binding protein is the observation that E2 core substantially depleted of p45 still retains its ability to fully bind E1 and function catalytically, but does not bind additional E3, while E2 core enriched with p45 binds additional E3. Whether the PDC from early larval stages contains an E3BP similar to that observed in yeast and mammals must await the purification of the PDC from these stages. Unfortunately, purification is complicated by the limited amounts of available tissue and the high protease activity found in the hatching fluids of those early larval stages.
Although the function of a novel E3BP in anaerobic ascarid mitochondria is still not completely clear, it appears that the binding of E3 to p45 may decrease the sensitivity of the complex to NADH inhibition. When a hybrid complex was constructed using the bovine kidney core, bovine kidney E1, and the ascarid E3, the hybrid complex had a significantly higher apparent K m for NAD ϩ and was more sensitive to NADH inhibition than the native ascarid complex. PDCs exhibit a wide range of sensitivities to NADH inhibition. For example, PDCs from facultative anaerobes, such as Escherichia coli, are very sensitive to NADH inhibition. As NADH accumulates during anaerobiosis, PDC activity is inhibited, which in turn, diverts pyruvate from the TCA cycle to fermentative pathways (33). In contrast, PDCs from anaerobes, such as Enterococcus faecalis, are much less sensitive to NADH inhibition (29). PDCs from higher eukaryotes also are less sensitive to NADH inhibition. In these organisms, PDC activity is regulated primarily by covalent modification and not end product inhibition. In addition, elevated NADH/NAD ϩ levels have a marked stimulatory effect on pyruvate dehydrogenase kinase activity which catalyzes the phosphorylation and inactivation of the complex (34,35). The PDC from adult A. suum muscle and other gut dwelling parasitic helminths is the most insensitive to NADH inhi-FIG. 5. Immunoblot of A. suum larval homogenates with antisera against the adult A. suum E2, p45, E1␣, and E1␤. Larval homogenates and the affinity purified antisera were prepared as described under "Experimental Procedures." E2 and p45 antisera were used at dilutions of 1:5000 and 1:2000, respectively. E1␣ and E1␤ antisera were both used at dilutions of 1:2000. UE, unembryonated eggs (150 g); L1 (150 g); L2 (150 g); L3 (80 g); and M, adult muscle PDC (1 g). *, rabbit IgG: L3 are recovered from rabbit lungs and homogenates often contain rabbit IgG. bition, and pyruvate dehydrogenase kinase activity in these complexes is also less sensitive to activation by NADH (7,17). Interestingly, when the amino acid sequence of the conserved redox active disulfide site of E3s from anaerobic parasitic helminths is compared to that of other E3s, a consistent sequence difference is observed. Phenylalanine is substituted for leucine at position 40 and a proline is located at position 38. These sequence alterations are conserved in E3s from anaerobic helminths representing two distinct helminth phyla, but not in the E3 from the aerobic free-living nematode, Caenorhabditis elegans. The significance of this sequence difference remains to be determined. The function of E3BP has been elucidated most directly in the S. cerevisiae PDC. Disruption of the gene encoding E3BP yields a catalytically inactive complex with a properly assembled E2 core and attached E1, but no E3, and suggests that while E3BP is required for E3 binding, it is not an integral part of the core or required for assembly (36). More important, deletion of most of the lipoyl domain of E3BP had no effect on PDC activity, suggesting that the lipoyl domain was not essential for E3 binding or E3BP function (36). While E3BP has a domain structure similar to E2, it has no catalytic activity, and lacks the HXXKG sequence near the carboxyl terminus proposed as part of the active site of all dihydrolipoamide acyltransferases (37). However, after the removal of the lipoyl domains of E2 by direct proteolysis or site-directed mutagenesis, it does appear that the lipoyl domain of E3BP can support the overall reaction of the complex, albeit at a greatly reduced rate (38,39). Indeed, the final specific activity of the PDC purified from A. suum muscle is significantly lower than that reported for PDCs from either yeast or mammals (7). Whether this reduced specific activity is related to the absence of a lipoyl domain on E3BP is not clear. Recently, an E3BP in the PDC of the insect trypanosomatid, Crithidia fasciculata, has been identified which appears to contain, not one, but multiple lipoyl domains (40). A similar situation has been described previously for E2s, where 1, 2, or 3 lipoyl domains have been identified, depending on the species, and there appears to be no physiological significance to the number of lipoyl domains in E2 (41). For example, site-directed mutagenesis of the three lipoyl domains of the E. coli E2 suggests that the inactivation of two of the three lipoyl domains does not affect catalysis (42). Whether more subtle regulatory properties are affected by these alterations has not been determined.