pyruvate dehydrogenase complex from the primitive insect trypanosomatid, crithidia fasciculata:...

ELSEVIER Molecular and Biochemical Parasitology 75 (1995) 87-97

MOLECULAR

il&mIcAL PARASITOLOGY

Rapid communication

Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl

dehydrogenase-binding protein has multiple lipoyl domains

Francisca Diaz*, Richard Komuniecki

Department of Biology, University of Toledo, Toledo, OH 43606-3390, USA

Received 26 June 1995; revision received 13 September 1995; accepted 13 September 1995

Abstract

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from the insect trypanosomatid, Crithidiufasciculuta, a member of the most primitive eukaryotic group to contain mitochondria. Sepa- ration of the purified PDC by SDS-PAGE yielded five bands of 70 (p70), 60 (p60), 55,46 and 36.5 kDa, which appeared to correspond to dihydrolipoyl dehydrogenase binding protein (E3BP), dihydrolipoyl transacetylase (E2), E3, Ela! and El& respectively. The purified complex did not exhibit endogenous PDH, kinase activity. p70 was much less abundant than ~60. Polyclonal antisera raised against p70 did not cross-react with ~60, and antisera raised against p60 did not cross-react with ~70, suggesting that p60 did not arise from p70 by proteolysis. Both p70 and p60 contained similar amino terminal sequences. Both sequences contained the MPALSP motif similar to sequences present in both E3BP and E2 from other sources. Incubation of the purified PDC with [2-‘4C]pyruvate in the absence of CoA resulted in the acetylation of both p70 and ~60, suggesting that both proteins contained lipoyl domains, but the specific incorporation of label into p70 was significantly greater than for ~60. Limited proteolysis of the acetylated complex with trypsin yielded two major fragments derived from p60 of 35 and 30 kDa, corresponding to E2, and E2,, and one major acetylated fragment of 58 kDa derived from ~70. Therefore, these results suggest that p70 is an E3BP and given its apparent M, and degree of acetylation, it contains multiple lipoyl domains.

Keywords: Pyruvate dehydrogenase complex; Crithidia fasciculata

Abbreviations: El, pyruvate dehydrogenase; E2, dihydrolipoyl transacetylase; E2a, subunit-binding domain of E2; E2,, catalytic domain of E2; E2,, lipoyl domain of E2; E3,

dihydrolipoyl dehydrogenase; E3BP, E3 binding protein (protein X); PDC, pyruvate dehydrogenase complex.

*Corresponding author, Tel.: +I 419 5302124; Fax: +I 419 5307737; E-mail: [email protected]

1. Introduction

Parasitic protozoa of the family Trypanosoma- tidae, order Kinetoplastida, include a number of

species of medical importance, including Trypanosoma cruzi, Trypanosoma brucei and

0166-6851/95/%09.50 0 1995 Elsevier Science B.V. All rights reserved SSDl 0166-6851(95)02498-3

88 F. Diaz. R. Komuniecki/Molecukw and Biochemical Parasitology 75 (199s) 87-97

several Leishmaniu spp. The kinetoplastids contain a single mitochondrion branching throughout the cell and, together with the euglenoids, are the most primitive eukaryotic group to contain mitochondria [ 1,2]. Therefore, they occupy a unique posi- tion in the evolution of eukaryotes. In the present study, we have characterized the pyruvate dehydrogenase complex (PDC) from the insect trypanosomatid, Crithidia fasciculata, in an at- tempt to better understand the origins of the eukaryotic-specific dihydrolipoyl dehydrogenase- binding protein (E3BP or protein X) identified in other eukaryotic PDCs.

The PDC contains three distinct catalytic components: pyruvate dehydrogenase (El), dihydrolipoamide transacetylase (E2) and dihydrolipoamide dehydrogenase (E3) [3-71. Multiple copies of E2 form the core. E2s contain three distinct domains: a variable number of lipoyl domains at the amino terminus (E2,_), a subunit-binding domain (E2s) and a catalytic domain (E2t) [8]. The evolutionary relationship between the number of lipoyl domains and the phylogeny of E2 is not apparent. For example, the Escherichia coli [9] and Azotobacter vinelandii [lo] PDCs contain three lipoyl domains, the Streptococcus faecalis [ 1 l] and mammalian PDCs [12] contain two lipoyl domains, and the Saccharomyces cerevisiae PDC [ 131 contains a single lipoyl domain. In prokaryotes, both El and E3 appear to bind directly to E2a, while in eukaryotes a specific E3-binding protein (E3BP) is present [14,15]. Exceptions to this generalization are the PDCs from anaerobic mitochondria of the parasitic nematodes, Ascaris suum and Parascaris equorum, which do not appear to contain an E3BP similar to that observed in other eukaryotic complexes [ 16,171. The site of E3 binding in these organisms has not yet been determined. E3BP has been cloned and sequenced from S. cerevisiae [ 151 and partial sequence is available from mammals [18-201. E3BP contains a domain structure similar to E2, including a single lipoyl domain, but does not exhibit transacetylase activity [21, 221. Studies with E3BP-deficient S. cerevisiae mutants indicate that, in the absence of E3BP, the E2 core assembles properly, but E3 does not bind (23,241.

In the present study, PDC has been purified to apparent homogeneity from C. fasciculata and its

subunit composition determined. The crithidial complex lacks intrinsic PDH, kinase activity and, in contrast to results reported for other eukaryotic PDCs, its E3BP appears to contain multiple lipoyl domains.

2. Materials and methods

2.1. Materials [2-‘4C]Pyruvate was purchased from New En-

gland Nuclear (Boston, MA) and [y-32P]ATP was from Amersham Co. (Arlington Heights, IL). Brain heart infusion was from Difco Laboratories (Detroit, MI). All electrophoresis reagents and molecular weight standards were obtained from Bio-Rad Laboratories (Richmond, CA) and reagents for immunoblotting were from Promega Corporation (Madison, WI). Immobiline@ DryStrips (pH 3-10) were obtained from Phar- macia Biotech (Uppsala, Sweden), urea from ICN Biomedicals, Inc. (Costa Mesa, CA) and Pluronic@’ F 68 from BASF Corporation (Parsip- pany, NJ). ProblottTM membranes were purchased from Applied Biosystems (Forest City, CA). Preblended ampholines and all other chemicals were of the highest grade available and obtained from Sigma Chemical Co. (St. Louis, MO). Crithidia fasciculutu was kindly supplied by Dr. Barbara Carter of the University of Toledo, Toledo, Ohio.

2.2. Culture of Crithidia fasciculata C. fasciculata were grown in 3.7% (w/v) brain

heart infusion (Difco), 20 mg 1-l hemin (prepared in 50 mM NaOH) and 20 mg 1-l gentamicin. The cultures were kept at 28°C with continuous agitation (200 rpm). After 48-60 h, cultures were harvested at a density of approximately lo* cells ml-’ by centrifugation at 7100 x g for 10 min at 4°C [25] and washed twice with phosphate- buffered saline (2.7 mM potassium chloride, 1.3 mM potassium phosphate monobasic, 137 mM sodium chloride and 6.5 mM sodium phosphate dibasic, pH 7.0). Final pellets were stored at -70°C prior to use.

2.3. Purification of the pyruvate dehydrogenase complex

All procedures were performed at 4°C unless

F. Diaz. R. Komuniecki/ Molecular and Biochemical Parasitology 7.5 (1995) 87-97 89

otherwise stated. Frozen C. fasciculata pellets (100 g wet weight) were resuspended in 200 ml of 100 mM potassium phosphate, 1 mM EDTA, 0.01 mM thiamin pyrophosphate, 2 mM dithiothreitol, pH 7.0, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 2.1 PM leupeptin, 3 PM aprotinin, 1 PM soybean trypsin inhibitor, 66 PM antipain, 152 PM e-amino caproic acid, 33 PM chymostatin and 29 PM pepstatin A (Buffer I). Cells were broken by two passages through a French Press at 16 000 psi, and the pH of the ho- mogenate was adjusted to 6.3 with 1.7 N acetic acid. After centrifugation at 18 000 x g for 25 min, the supematant was filtered through cheesecloth (supematant), brought to room temperature, and 0.048 volumes of 35% (w/v) polyeth- ylene glycol (PEG) were added dropwise with constant stirring. After 15 min, the mixture was centrifuged at 14 000 x g for 15 min at 4°C. The PDC activity remaining in the supematant was precipitated by the addition of 0.12 volumes of 35% (w/v) PEG and stirred for 30 min on ice. After centrifugation at 25 000 x g for 10 min, the pellet was resuspended in 50 ml of Buffer I and stored overnight at 4OC (PEG II). The sample then was homogenized with a Teflon-pestle tissue homogenizer and pH adjusted to 6.2 with 1.7 N acetic acid. After stirring for 15 min, the mixture was centrifuged at 14 000 x g for 30 min. The supematant was warmed to room temperature and 0.06 volumes of 35% (w/v) PEG were added. After stirring for 30 min, the mixture was centrifuged at 25 000 x g for 10 min. At this point, most of the PDC activity precipitated, while cr-ketoglutarate dehydrogenase complex activity remained in the supematant. The pellet was resuspended in 40 ml of Buffer I and stored on ice for 1 h (PEG III). Following an initial clarification at 14 000 x g for 40 min (PEG III clarified), the supematant was centrifuged again at 70 000 x g for 25 min to remove insoluble material. This supematant was layered on top of a 2.5 ml cushion of 15% (w/v) sucrose in Buffer I and centrifuged at 155 000 x g for 2 h. The pellet was resuspended overnight in 8 ml of Buffer I containing 30% (w/v) glycerol (Buffer II; Ultracentrifugation I). 4fter homogtiuzatlon with a Teflon-pestle tissue homogenizer, the sample was clarified at 14 000 x g for 40 min, and the supematant (Ultracentrifugation I

clarified) was centrifuged at 70 000 x g for 25 min to remove insoluble material. This supematant was centrifuged at 155 000 x g for 2 h. The final pellet was resuspended in 1 ml of Buffer II (Ultracentrifugation II). The PDC was stored at -20°C and the activity was stable for at least 3 months. In some preparations, insoluble material formed during the third PEG precipitation and the final pellet did not yield a pure PDC. In this case, further purification on Sepharose CL-2B and hydroxylapatite was required, as outlined below.

2.4. Additional chromatographic steps When PDC preparations were not homo-

geneous after PEG precipitation (as judged by SDS-PAGE), the sample was microfuged and applied to a Sepharose CL-2B column (1.5 x 80 cm) equilibrated in 100 mM potassium phosphate, 1 mM EDTA, 1 mM benzamidine, 2.1 PM leupeptin, pH 7.0. The flow rate was 15 ml h-’ and 4-ml fractions were collected. Fractions containing PDC activity were centrifuged at 155 000 x g for 5 h. Pellets were resuspended in Buffer II and applied to an hydroxylapatite column (0.9 x 2 cm) equilibrated in 100 mM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM thiamin pyrophosphate, 1 mM benzamidine, 2.1 PM leupeptin, pH 7.0. The column was washed with 200 mM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM thiamin pyrophosphate, 1 mM benzamidine, 2.1 pM leupeptin, pH 7.0 (Buf- fer III), and PDC activity was eluted with Buffer III containing 6% (w/v) ammonium sulfate. Frac- tions containing PDC activity were centrifuged at 155 000 x g for 5-6 h. Final pellets were resuspended in Buffer II containing protease inhibitors and stored at -20°C.

2.5. Protein determination Protein was determined according to Bradford

[26]. Bovine serum albumin was used as a standard.

2.6. PDC assay PDC activity was determined spectro-

photometrically at 340 nm following NADH for- mation as described previously [27]. The reaction

mixture contained 50 mM potassium phosphate, pH 7.4, 1 mM MgC12, 0.2 mM NAD+, 2 mM

90 F. Diaz, R. Komuniecki /Molecular and Biochemical Parasitology 75 (1995) 87-97

dithiotreitol, 0.1 mM thiamin pyrophosphate, 0.1 mM CoA, 4 mM pyruvate and enzyme in a final volume of 1 ml. After a 2-min preincubation, the reaction was initiated by the addition of pyruvate. All kinetic analyses were performed with the non- linear regression data analysis program ENZFIT- TER (Elsevier/Biosoft). Simple weighting was used to fit the data to the Michaelis-Menten equation. The apparent K,,, for CoA was determined using a CoA-regenerating system containing citrate syn- thase and oxaloacetate as described previously

WI.

2.7. Gel electrophoresis and immunoblotting Samples were separated on 10% SDS-

polyacrylamide gels according to Laemmli [29] and proteins were visualized by staining with Coomassie brilliant blue R-250. Densitometric scanning of stained gels and autoradiographs was carried out with an Isco model 1312 gel scanner. To determine the relative amounts of each protein, the areas under the peaks were excised and weighed.

For immunoblotting, samples were transferred to nitrocellulose with a constant current of 400 mA for 3-4 h in 25 mM Tris-HCl, 192 mM glycine, 20% (v/v) methanol. To detect bands of interest, each specific antisera was used in conjunction with the Promega kit, ProtoBlot@ Western blot AP system, using goat anti-rabbit IgG secondary anti- body conjugated to alkaline phosphatase (AP). The procedure was performed as described by the manufacturer.

For two-dimensional gel electrophoresis, iso- electric focusing (IEF) was performed in 1 l-cm precast Immobiline@ DryStrips (Pharmacia Biotech) containing an immobilized pH gradient from 3-10. Immobiline strips were rehydrated overnight in 8 M urea, 0.5% (w/v) Triton X-100, 0.2% (w/v) dithiothreitol, 0.5% (w/v) Nonidet P- 40. C. fasciculata PDC (50 pg) was solubilized in 9 M urea, 1% (w/v) dithiothreitol, 2% (v/v) preblended ampholines, pH 3.5-9.5 (Sigma), 2% (w/v) Nonidet P-40 and bromophenol blue in a final volume of 50 ~1. IEF was performed in the rehydrated Immobiline@ DryStrips using a Multiphor II electrophoresis system (Pharmacia Biotech) by running at 150 V for 30 min, 300 V for

5 h and 1150 V for 17.5 h. After a total of 21 700 Vh, Immobilines DryStrips were equilibrated in 50 mM Tris-HCl, pH 6.8, 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 1% (w/v) dithiothreitol and bromophenol blue. Equilibration of the strips was performed with gentle agitation for 15 min (2 x ). For the second dimension, the Immobiline@ DryStrips were placed on top of a 10% gel and separated by SDS-PAGE as described previously t291.

2.8. Acetylation of the pyruvate dehydrogenase complex with [2-“Cjpyruvate

Purified PDC was acetylated with [2- 14C]pyruvate in the absence of CoA as described previously [17] with the exception that only 6900 cpm nmol-’ of [2-i4C]pyruvate were used and autoradiography was performed by exposing the dried gel to a Kodak BioMax MR film.

2.9. Limited proteolysis of acetylated complex with trypsin

Acetylated PDC was prepared as described above and instead of stopping the reaction with the sample buffer, the acetylated complex (320 pg) was diluted in 4 ml of 50 mM MOPS, pH 6.5, and centrifuged at 155 000 x g for 4 h to exchange buffers and remove the protease inhibitors present in the acetylation reaction. The PDC pellet was resuspended in the same buffer to a protein con- centration of 1 mg ml-‘. The acetylated complex was incubated with 6.25% (w/w) trypsin (1 mg ml-’ stock solution prepared in 50 mM MOPS, pH 6.5, and stored at -20°C) at 4°C for 1 h. The reaction was stopped as described above.

2. IO. Superose 12 chromatography in the presence of2MKBr

The purified crithidial PDC (1.5 mg) was diluted in 2.5 ml of 50 mM potassium phosphate, pH 7.0, and centrifuged at 155 000 x g for 3.5 h. The resulting pellet was resuspended in 0.3 ml of 50 mM potassium phosphate, 2 M KBr, 10 mM 2- mercaptoethanol, 2 mM EDTA, 0.2 mg ml-’ Pluronice F 68, pH 7.0, and incubated for 30 min at room temperature. The solution was then frozen and stored for 30 min in liquid nitrogen [30]. After thawing, the sample was microfuged for 5 min and

F. Dioz, R. Komuniecki/ Molecular and Biochemical Parasitology 75 (1995) 87-97 91

separated by FPLC on a Superose 12 HR 10130 column (Pharmacia Biotech) equilibrated in the same buffer. The flow rate was 0.2 ml min-’ and OS-ml fractions were collected. Peak fractions were pooled. The first peak was centrifuged at 155 000 x g for 6 h and the pellet was resuspended in 0.2 ml of 50 mM potassium phosphate, pH 7.0. The other peaks were concentrated to about 0.2 ml, in a Centricon 30, with several changes of 50 mM potassium phosphate. Peaks were analyzed by SDS-PAGE. The column was calibrated under the running conditions described above using Blue dextran (2000 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa) and car- bonic anhydrase (29 kDa) as molecular weight standards.

with the band homogenates, without adjuvant and, a week later, blood was drawn from the mid- dle vein of the ear and serum stored at -20°C. Serum titers were determined by immunoblotting. If serum titers were low, rabbits were boosted every 2 weeks until the desired titers were reached.

2.13. Determination of amino terminal sequences The amino terminal sequences of proteins of in-

terest were determined as described previously

1171.

3. Results

3.1. Purification of the pyruvate dehydrogenase complex

2.11. Phosphorylation of PDC with [T-~~P]ATP Purified PDC was incubated with [T-~~P]ATP

as described previously [17] and PDH, kinase activity was determined as described in [28].

2.12. Preparation of antisera The purified PDC was separated by SDS-

PAGE, stained with Coomassie blue, and the bands of interest were excised and washed several times in distilled water and 50 mM sodium bicarbonate. The washed bands were homogenized with a Teflon-pestle tissue homogenizer with 50 mM sodium bicarbonate. The specific band homogenates were mixed with an equal volume of Freund’s Complete Adjuvant and injected subcutaneously in several sites of the back of New Zealand rabbits type SPF. After 3 weeks, rabbits were boosted

The PDC has been purified to apparent homogeneity from the insect trypanosomatid, C. fasciculata, using a combination of PEG precipita- tions and centrifugations. The results of a typical purification are outlined in Table 1. In some preparations, the PDC was essentially homo- geneous after the final PEG precipitation. In other preparations, additional clarification steps were required after the third PEG precipitation to remove insoluble material, and additional chromatographic steps on Sepharose CL-2B and hydroxylapatite were required to achieve homogeneity. The reasons for this variability are unclear, but it probably results in part from a large precipitate that formed in initial clarification steps which interfered with PEG precipitation. In any event, the PDCs prepared using either protocol were identical. The presence of the protease inhibi-

Table 1 Purification of the pyruvate dehydrogenase complex from C. fasciculata

Step Volume

(ml) Protein (mg)

Activity (pm01 min-‘)

Specific activity (pm01 min-’ (mg protein)-‘)

Yield

(“W

Supematant 242 3722 757 0.2 -

PEG II 59 1502 933 0.6 100 PEG III 40 550 821 1.5 88 PEG III= 35 335 680 2.0 13 Ultracentrifugation I 8.5 75 391 5.2 42 Ultracentrifugation Ia 7.8 39 368 9.4 39 Ultracentrifugation II 1 12 217 22.1 30

aPellet was resuspended and clarified by centrifugation at 14 000 x g for 40 min.

F. Diar. R. Komwriecki/ Molecukn and Biochemical Parasitology 75 (1995) 87-97

- 97

- 50

- 35

Fig. I. One- and two-dimensional electrophoresis of the purified C. farciculata PDC. (A) SDS-PAGE of the purified C. fasciculata PDC (10 pg) on a 10% (w/v) gel. (B) TWO- dimensional gel electrophoresis: IEF of the crithidial PDC (50 rg) was performed in Immobilinee DryStrips, pH 3-10 (Phar- macia Biotech), as described in Materials and methods. Focus- ing was performed at 150 V for 30 min, 300 V for 5 h and I I50 V for 17.5 h. Following IEF, SDS-PAGE on a 10% (w/v) gel was performed in the vertical direction. Positive indicates the acidic end and negative indicates the basic end of the IEF. Molecular weight markers are noted at the right.

tor cocktail throughout the purification was necessary to to maximize PDC recovery. When the purification was performed in the presence of only PMSF, benzamidine and leupeptin, yields were much lower and the enzyme preparations showed additional bands after SDS-PAGE. The final specific activity of the C. fasciculata PDC varied from 20 to 30 pmol min-’ (mg protein)-’ and was com- parable to specific activities reported for the yeast

complex [31]. These purified PDC preparations were free of ar-ketoglutarate dehydrogenase complex activity, which copurified with PDC until the final PEG precipitation.

3.2. Subunit composition Separation of the C. fasciculata PDC by either

SDS-PAGE or two-dimensional gel electrophoresis yielded four major bands of 60 (p60), 55 (p55), 46 (p45) and 36.5 (~36.5) kDa and a less intensely staining band at 70 kDa (~70; Fig. 1). Densitometric analysis of one-dimensional gels indicated that the area of the peak corresponding to p60 was about 5 times greater than the area corresponding to ~70, and that the proportion of all five bands was similar in all preparations (n = 6; data not shown). Initial attempts to identify the in- dividual subunits of the complex involved separation by SDS-PAGE, transfer to ProblottTM and amino terminal sequencing. The amino terminal sequences are summarized in Table 2. The sequences for ~55, p46 and ~36.5 exhibited signiti- cant similarities to E3, Elan and El/3 from other organisms, respectively. Both p70 and p60 contained similar amino terminal sequences and both sequences contained the MPALSP motif present in both E3BP and E2 from other sources (Table 2).

3.3. Acetylation of the PDC with [2-‘4Clpyruvate and identification of E3BP

To gain more insight into the identity of p70 and ~60, the purified complex was incubated with [2- “C]pyruvate. Incubation of the PDC with [2-

Table 2 Amino terminal sequences of the purified C. fasciculata PDC subunits and comparison with E2 and E3BP sequences from other

organisms

Pa

P70 S. cerevisiae

E2 E3BP

Bovine heart E2 E3BP

P55

p46 ~36.5

LTITPIPMPALSPTMEKGKI Present study VNFEAVF. a.. . . Present study

ASYPEH.1.G. ...... .TQ.NL I131 AVKTFS . ..M.. ..... G. I151

SLPPHEKV.L.S.. . - -QA.T- [I81 ADPIK.L..S . . . . . . E.N. 1191

ASYDVTVIGGGPGGGYVAAIKAAQLGLKA Present study ATKTVPLKPPHPFKLHAA Present study ATTNMTVRDAIXXALDEEIA Present study

Dots indicate identity with the p60 sequence.

F. Diaz, R. Komuniecki/Molecular and Biochemical Parasitology 75 (1995) 87-97 93

A B C M, (x 10-3)

p70 - p60 - E3 -

Ela -

Elfi - - 35

- 30

1 2 34 56 78

Fig. 2. SDS-PAGE of intact and trypsindigested PDC and identification of p70 and p60 by autoradiography and immunoblotting. Acetylation of the purified PDC with [2- “C]pyruvate and limited proteolysis of the acetylated complex with trypsin was performed as described in Materials and methods. Intact and trypsin-digested, acetylated PDCs were separated by SDS-PAGE in replica gels (10% SDS- polyacrylamide gels). Staining with Coomassie blue (A), autoradiography (B) and immunoblotting (C) were performed as described in Materials and methods. Lanes I, 3, 5 and 7, intact acetylated PDC (20, 20, 2 and I cg protein, respectively). Lanes 2,4,6 and 8, trypsin-digested acetylated PDC (20,20, 2 and 1 pg protein, respectively). Lanes 5 and 6, immunoblot with polyclonal antisera against p70 (1:lOOO). Lanes 7 and 8, immunoblot with polyclonal antisera against p60 (1: 1000).

14C]pyruvate, in the absence of CoA, results in the acetylation of lipoyl domains [16,22]. When the C. fusciculata PDC was incubated with [2- “C]pyruvate, acetylation of both p70 and p60 was observed (Fig. 2B, lane 3). Densitometric analysis of the autoradiogram or direct counting of the acetylated bands indicated that p60 exhibited only about twice as much incorporation of radiolabel as p70 (data not shown). This data along with the apparent molecular weight observed after SDS-PAGE suggests that p70 contains multiple lipoyl domains. Alternatively, p60 might have arisen as a proteolytic degradation product of p70 by the selective removal of a lipoyl domain, and the C. fasciculata PDC might not contain E3BP, as has been observed for PDCs from parasitic nematodes [ 16,171. However, this possibility is not likely, since the crithidial PDC

was purified in the presence of protease inhibitors, and its final specific activity was similar to that reported for the yeast complex. To further exclude this possibility, specilic polyclonal antisera were prepared against both p60 and ~70. Antisera prepared against p70 did not cross-react with ~60, and the antisera against p60 did not cross-react with p70 on immunoblots of the purified C. fusciculutu PDC (Fig. 2C, lanes 5 and 7). In addition, the antisera against p70 recognized E3BP, and to a much lesser degree E2, on immunoblots of dehydrogenase-deficient bovine kidney PDC supplied by Dr. Thomas E. Roche, Kansas State University (data not shown). The reason for the slight cross-reactivity of the p70 antisera with E2 probably results from the presence of lipoyl domains in both proteins. In fact, at much higher titers the antisera to p70 also cross-reacted weakly with p60 (data not shown). These results strongly suggest that p70 and p60 are different proteins, and that p70 is E3BP.

To gain further insight into the domain structures of both p70 and ~60, the intact, acetylated crithidial complex was partially digested with trypsin (Fig. 2A, lane 2) followed by autoradiography and immunoblotting (Fig. 2B, lane 4; 2C, lanes 6 and 8), as has been described for the E2 domain mapping of the bovine kidney and A. suum PDCs [16,21]. p60 was degraded to an acetylated fragment of about 35 kDa, which corresponded to E2,_, and a fragment of about 30 kDa, which corresponded to E2, (Fig. 2, lanes 4 and 8). The breakdown of p70 was more complex, yielding a major acetylated fragment at 58 kDa and a minor, acetylated, doublet at 65-66 kDa (Fig. 2, lanes 4 and 6). Interestingly, the 58kDa fragment had the same amino terminal sequence as p70, suggesting that proteolysis occurred at the carboxyl terminus.

3.4. Partial resolution of the purified complex Attempts to resolve the complex using techni-

ques described for other PDCs met with only partial success. Resolution in 2 M NaCl, followed by chromatography on Superose 12 dissociated a por- tion of both El and E3 (about 50%), but left substantial amounts of both dehydrogenase subunits still bound to the core (data not shown). More interestingly, resolution in 2 M KBr, follow-

94

0.8

0.6

F. Diaz, R. Komtmiecki/Molecular and Biochemical Parasitology 75 (1995) 87-97

p70 -

Pa -

E3 -

Ela -

ElP -

” 2 12 16 20 24 28 32

Fraction

Fig. 3. Partial resolution of the PDC in the presence of 2 M KBr. The purified crithidial PDC (1.5 mg) was incubated for 30 min in 0.3 ml of 50 mM potassium phosphate, 2 M KBr, 10 mM 2-mercaptoethanol, 2 mM EDTA, 0.2 mg ml-’ Pluronic@ F 68, pH 7.0, as described in Materials and methods. The sample (0.2 ml) was applied to a Superose 12 column equilibrated in the same buffer. The flow rate was 0.2 ml min-’ and 0.5-m] fractions were collected. Insert: SDS-PAGE, on a IO?/0 (w/v) SDS-polyactylamide gel, of the pooled peak fractions; *C. faxicufata PDC; 1 (fractions 14-17); II (fractions 20-22); III (fractions 23-25) and IV (fractions 26-28). Molecular weight markers of 64, 50 and 36 kDa, are noted at the right.

ed by chromatography on Superose 12 yielded four distinct peaks (Fig. 3). Peak I eluted with the void volume and contained p70-p60-Ela (and some El&; peak II migrated at about 190 kDa and

contained E3 and a p70 degradation product, which corresponded to the 6%66-kDa doublet observed during partial proteolysis of the acetylated complex with trypsin (Fig. 2, lane 4); peak III migrated at about 100 kDa and contained E3, and peak IV migrated at about 38 kDa and contained Elfi (Fig. 3). The selective removal of El/3 from the core without the loss of Ela suggests that Elan may play a role in the binding of El to the core, although the possibility that an individu- al El/3 monomer was removed from the &I2 El tetramer cannot be excluded.

3.5. Kinetic properties The kinetic parameters of the purified C.

fasciculuta PDC are summarized in Table 3. These values are similar to those reported for mammalian PDCs, with the exception of the elevated apparent K,,, for pyruvate, which was also observed in the yeast complex [31]. GTP, GDP, GMP, ATP, ADP and AMP at 1 mM did not inhibit PDC activity when assayed at either 0.4 and 4 mM pyruvate. In addition, alanine, proline, glutamate, aspartate, malate, citrate and isocitrate at 1 mM had no effect on PDC activity when assayed as described above.

To determine if the C. fasciculutu PDC was regulated by phosphorylation-dephosphorylation, the purified complex was incubated with [r- 32P]ATP. No inactivation of PDC activity or incorporation of radioactivity into Elar was observed (n = 3, data not shown). In mammalian PDCs, PDH, kinase is tightly associated with the lipoyl domains of E2 and does not readily dissociate during purification 1321. However, it is possible that the crithidial PDH, kinase was not as tightly associated and was lost during puritica-

Table 3 Kinetic properties of the PDC from different organisms

Organism

Mammalian A. suwn

S. cerevisiae C. fasciculata

Apparent K,,, (mM) Reference

Pyruvate CoA NAD+

15-60 S-10 40 1391 200-400 0.5 10 1281 650 14 72 1311 300-500 10 60 Present study

F. Diaz, R. Komunieckiihfolecuiar and Biochemical Parasirotogv 75 (1995) 87-97 95

2 a

PC 4 0.02 I 1 I

0 10 20 30 40

Minutes

Fig. 4. Effect of ATP on PDC activity. The purified crithidial PDC (120 pg) was incubated in duplicate at 30°C in 50 mM MOPS, 60 mM KCI, 3 mM dithiothreitol, 2 mM MgCI,, I mM ATP, pH 7.4, in a final volume of 1 ml. After 10 min (arrow), purified A. suum PDC (2.3 mg) was added to one sample and total PDC activity was assayed at the times indicated. Open circles, C. fasciculata PDC; closed circles, C. fascicufata plus A. suum PDCS.

tion. Therefore, to examine this possibility, partially purified preparations of the crithidial PDC also were assayed for kinase activity. No phosphorylation or inactivation was observed (data not shown). Finally, the purified C. fuscicufatu PDC was incubated with the purified A. suum PDC containing endogenous PDH, kinase activity and [r- 32P]ATP (Fig. 4). PDC activity decreased to levels approaching those of the initial crithidial activity, suggesting that the ascarid complex was nearly completely inactivated. In addition, no phosphorylation of the crithidial Ela! was observed in these incubations after analysis by SDS-PAGE and autoradiography. These results suggest that the crithidial complex lacks PDH, kinase activity, at least under the growth conditions used in the present study.

4. Discussion

The PDC has been purified to apparent homogeneity from the primitive insect

trypanosomatid C. fasciculata. These organisms contain a single, highly-branched mitochondrion with a specialized region, the kinetoplast 1331. Together with the euglenoids they constitute the most primitive eukaryotes to contain mitochondria and, therefore, are excellent organisms for evolutionary studies [ 1,2]. Crithidia fasciculata normally resides in the mosquito gut, where carbo- hydrate levels are low, but it maintains a rate of glucose consumption almost 10 times that observed in mammalian tissues [34]. Interestingly, even in the presence of oxygen, it excretes a variety of reduced organic products in common with many other parasites [34-361. Not surprisingly, PDC activity is high in this organism and for this reason it was chosen to examine the evolutionary origins of the eukaryotic specific E3-binding protein, which had previously been identified in the evolutionarily more advanced S. cerevisiae, and in mammals [14,15].

The crithidial PDC has a subunit composition similar to that reported for the yeast PDC, except that the crithidial E3BP appears to contain multiple lipoyl domains. Each subunit of the crithidial complex was tentatively identified by amino terminal sequencing and, in contrast to results reported for the yeast and mammalian PDCs, amino terminal sequence could be obtained for all subunits of the crithidial complex. Two subunits, p70 and ~60, contained the MPALSP motif characteristic of E2 and E3BP from other organisms [13,15,18,19]. While p60 was much more abundant than ~70, both subunits were readily acetylated after incubation in [2- “C]pyruvate, and the specific incorporation of label into p70 was substantially greater than into ~60. Immunoblotting with subunit-specific polyclonal antisera indicated that p60 did not arise from the proteolytic degradation of ~70. Taken together, these data suggests that p70 is E3BP and that it contains multiple lipoyl domains, although the ultimate proof of this hypothesis must await the cloning and sequencing of ~70. Most important, it will be of interest to determine if p70 possesses dihydrolipoyl transacetylase activity. The physiological significance of multiple lipoyl domains in E3BP is unclear. E2 contains from one to three lipoyl domains depending on the source

96 F. Diaz. R. Komuniecki/ Molecular and Biochemical Parasitology 75 (1995) 87-97

and there appears to be no physiological significance to the number of lipoyl domains [9- 131. In fact, site-directed mutagenesis of the three lipoyl domains of the E. coli E2 suggests that the inactivation of two of the three lipoyl domains has no obvious effect on catalysis [37]. Whether there are more subtle effects on regulation remains to be determined.

Crithidia fasciculata is closely related to a number of other trypanosomatids of medical importance, including T. brucei and T. cruzi [ 1,381, and it is well documented that antibodies raised against C. fmciculata proteins often cross-react with their homologs in other trypanosomes [25]. Therefore, with the development of subunit-specific antisera in the present study, insights into the regulation of this important complex during the transition from bloodstream to insect forms of other trypanosomatids should now be possible, especially as they relate to the regulation of PDC expression during the mitochondrial biogenesis in these medically relevant pathogens.

Acknowledgements

We would like to thank Michele Klingbeil for performing the kinase assays. This work was sup- ported by National Institute of Health Grant AI 19427 to RWK.

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pyruvate dehydrogenase complex from the primitive insect trypanosomatid, crithidia fasciculata:...

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