©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stage-specific Isoforms of Complex II (Succinate-Ubiquinone Oxidoreductase) in Mitochondria from the Parasitic Nematode, Ascaris suum(*)

(Received for publication, September 22, 1994; and in revised form, November 2, 1994)

Fumiko Saruta Toshiaki Kuramochi (§) Kayako Nakamura Shinzaburo Takamiya (1) Yong Yu (1) Takashi Aoki (1) Kazuhisa Sekimizu (2) Somei Kojima Kiyoshi Kita (¶)

From the  (1)Department of Parasitology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108 Japan, the Department of Parasitology, Juntendo University, School of Medicine, Tokyo, Japan, and the (2)Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Complex II from mitochondria of the adult parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron transport observed in these organelles. In contrast, mitochondria isolated from free living second stage larvae (L2) of A. suum show much lower fumarate reductase activity than those from adults, whereas succinate dehydrogenase activities of mitochondria in both stages are comparable. In the present study, biochemical and antigenic properties of the partially purified enzymes from both larval and adult mitochondria were compared. Larval complex II eluted from the DEAE-Cellulofine column chromatography at a lower salt concentration than adult enzyme, whereas the apparent molecular size of both enzyme complexes estimated by gel permeation column chromatography was the same. The fumarate reductase activity of larval complex II was less than 3% of that of adult enzyme, and the K values for substrates were significantly different between the two complexes. The flavoprotein subunit of larval complex II could be distinguished from that of adult complex II by two-dimensional gel electrophoresis and peptide mapping. The antibody against the smallest subunit (small subunit of cytochrome b) of the adult enzyme did not cross-react with that of the larval enzyme.

These results suggest that larval complex II differs from adult enzyme and is more similar to aerobic mammalian enzymes with low fumarate reductase activity. This is the first direct indication of the two different stage-specific forms of mitochondrial complex II.


INTRODUCTION

The adult parasitic nematode, Ascaris suum, resides in the host small intestine, where oxygen tensions are low, and has exploited a unique anaerobic mitochondrial respiratory chain as an adaptation to its microaerobic habitat. In this anaerobic respiratory chain, the reducing equivalents from NADH are transferred to two enzyme systems, the fumarate reductase of complex II (succinate-ubiquinone oxidoreductase) and electron-transfer flavoprotein rhodoquinone oxidoreductase, via rhodoquinone(1, 2, 3, 4) . Electron transfer from NADH to fumarate or enoyl CoA is coupled to ATP synthesis by a site I phosphorylation in complex I (NADH-ubiquinone oxidoreductase). It should be stressed that unlike the mammalian enzyme, complex II of adult A. suum functions in the reverse direction, as a fumarate reductase rather than as a succinate dehydrogenase(5, 6, 7) . In contrast to findings for the adult nematode, oxygen is required for larval development, and the ratio of succinate dehydrogenase to fumarate reductase in the fertilized egg (1.05) is intermediate between that of the adult (0.05) and mammals (20-30)(7) . Recently, we have found the respiratory chain of the mitochondria isolated from free living second stage larvae (L2) has substantial cytochrome oxidase activity and is similar to that of the aerobic mammalian host. However, the succinate dehydrogenase/fumarate reductase ratio of larval mitochondria (0.87) decreases at latter stages of development(8) . The change in the succinate dehydrogenase/fumarate reductase ratio during the life cycle suggests the two isoforms of complex II exist in A. suum. Different antigenic properties of larval mitochondria against anti-adult complex II antibodies support this idea(8) .

Complex II is generally composed of four polypeptides and appears to be highly conserved(9, 10) . The largest flavoprotein subunit (Fp) (^1)of 70 kDa contains covalently bound FAD. The second-largest, 30-kDa subunit (Ip) contains three different types of iron-sulfur center. The Fp and Ip subunits form the catalytic portion of complex and transfer reducing equivalents from succinate to water-soluble dyes, such as 2,6-dichlorophenol indophenol, or from reduced methylviologen to fumarate. Two small hydrophobic membrane-anchoring polypeptides with molecular masses of about 15 and 13 kDa (cytochrome b; cybL and cybS) seem to be essential for the interaction between the complex and quinone species. In Escherichia coli, succinate dehydrogenase is synthesized during aerobic growth, whereas fumarate reductase is induced in anaerobic culture. The genes for both enzyme complexes have been cloned (11, 12, 13, 14) , and their gene products have been purified and well characterized(15, 16) . We have isolated cDNAs for the Fp subunit of complex II from adult A. suum and free living nematode, Caenorhabditis elegans(17) . The amino acid sequences for the Fp subunits from both nematodes were quite similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme as a succinate dehydrogenase. However, no direct evidence for the presence of two distinct enzymes succinate dehydrogenase and fumarate reductase in the mitochondria of the same organisms has been obtained.

In the present study, complex II was isolated from larval and adult A. suum mitochondria under the same conditions, and biochemical and antigenic properties were compared to investigate the possible existence of two different forms of complex II.


MATERIALS AND METHODS

Isolation of Mitochondria from A. suum

Mitochondria from adult A. suum muscle and second stage larvae (L2) were prepared as described previously(8) . Succinate dehydrogenase and fumarate reductase activities were measured at 25 °C(17) . The specific activity of succinate dehydrogenase was always more than 0.15 µmol/min/mg of protein in both mitochondria.

Extraction and Separation of Complex II from A. suum Mitochondria

For separation by gel permeation, mitochondria (1 mg) were solubilized with the ionic detergent Sarkosyl (3% w/v), and the complex II was separated by high performance liquid chromatography (Shimadzu LC-10A) on TSK gel-G3000SW column (Tosoh) as described previously(18) . The elution of proteins and cytochromes was monitored by absorbance at 280 and 412 nm, respectively. For separation by ion exchange column, mitochondria (8 mg) were solubilized with the non-ionic detergent sucrose monolaurate (2.5% w/v) in 10 mM Tris-HCl (pH 7.6). After centrifugation at 200,000 times g for 60 min, the supernatant was applied to a column of DEAE-Cellulofine (1.3 times 3.0 cm). The column was washed with 0.1% (w/v) sucrose monolaurate, 10 mM Tris-HCl (pH 7.6), and then complex II was eluted with the same buffer containing a 100-ml linear gradient of 0-0.15 M NaCl at a flow rate of 30 ml/h. All conditions were exactly the same for both the larval and adult complex II.

Two-dimensional Gel Electrophoresis and Western Blotting Analysis

Two-dimensional gel electrophoresis was performed according to O'Farrell (19) with slight modification. Nonequilibrium pH gradient electrophoresis was used in the first dimension. The gel composition was 3% acrylamide, 9.2 M urea, 4% (v/v) Ampholine (pH 3-10), and 2% (v/v) Nonidet P-40. Proteins were solubilized from mitochondria with 2.5% (w/v) sucrose monolaurate and were precipitated with 5% (w/v) trichloroacetic acid. The precipitant was suspended with lysis buffer containing 9.5 M urea, 2% (v/v) Ampholine, 2% (v/v) Nonidet P-40, 5% (v/v) 2-mercaptoethanol and then loaded and run at 300 V for 3 h. Second dimensional gel electrophoresis in the presence of SDS and subsequent analysis by Western blotting were performed as described previously (7, 8) using monoclonal antibody against adult Fp and polyclonal antibodies against adult Ip and cybS.

Peptide Mapping by Limited Proteolysis

Proteolytic digestions were performed according to Cleveland et al. (20) with slight modification. Extracts from A. suum with 2.5% (w/v) sucrose monolaurate were concentrated by precipitation with 5% (w/v) trichloroacetic acid. Proteolytic digestions were performed in the 50 µl of reaction mixtures containing 50 mM Tris-HCl (pH 8.0) (for trypsin) or 50 mM ammonium bicarbonate (pH 8.0) (for V8 protease) at 37 °C. After the addition of 2-mercaptoethanol and SDS to final concentrations of 5 and 2%, proteolysis was stopped by boiling for 2 min. Samples were loaded to the 15% polyacrylamide gel, and then the electrophoresis and Western blotting were performed as described previously(7, 8) .

Other Methods

Spectrophotometric measurement at room temperature was performed with a Shimadzu UV-3000 dual wavelength spectrophotometer. Protein was determined according to Lowry et al. (21) with bovine serum albumin as the standard.


RESULTS

Separation of Complex II by Column Chromatography

Complex II has been purified from adult A. suum muscle mitochondria (5) and well characterized as a fumarate reductase(6, 7) . However, the protocol is not applicable to the purification of complex II from L2, not only because of the limited amount of starting material, but also because of the coexistence of other electron transfer complexes, i.e. complex III (ubiquinol-cytochrome c oxidoreductase) and complex IV (cytochrome c oxidase) in the L2 mitochondria. Therefore, to investigate differences between complex II of the L2 and adult, elution profiles of complex II were analyzed during gel filtration and ion exchange chromatography. For gel filtration, a microanalytical system, which we have established for the separation of respiratory complexes from bacterial and mitochondrial membranes(6, 18) , was used. Both larval and adult mitochondria were solubilized by anionic detergent, Sarkosyl, and the complexes were separated on TSK gel-G3000SW in the presence of Sarkosyl. The elution of complex II was monitored by measuring succinate dehydrogenase activity, and a similar peak of activity was observed for both complexes (Fig. 1). In contrast to succinate dehydrogenase activity, fumarate reductase activities of both complexes were different. Complex II from the adult showed high fumarate reductase activity, whereas complex II from the L2 did not. The retention time (17 min) corresponded to an apparent molecular mass of 120 kDa. Thus, both the larval and adult complex II appear to have the same size, and to be monomers composed of four polypeptides (70, 30, 15, and 13.5 kDa). The elution of cytochromes was monitored by absorption at 412 nm (Soret band), and the elution position of the main cytochrome peak was identical to that of succinate dehydrogenase activity (Fig. 1A). This absorbance peak is derived from succinate-reducible cytochrome b in the adult complex II, as shown previously(6) . Similar to adult complex II, cytochromes also eluted at the same retention time in the L2 (Fig. 1B), indicating that the L2 complex II also has a cytochrome b component. It is worthy of note that the Soret absorption peak of the oxidized form of cytochrome b in L2 complex II was at shorter wavelength than that of adult complex II as discussed later.


Figure 1: Elution profiles from HPLC of A. suum complex II. Complex II was solubilized with 3% (w/v) Sarkosyl and separated on a gel permeation column (TSK gel-G3000SW) as described under ``Materials and Methods.'' One mg of solubilized protein of adult (A) and L2 (B) was applied in each case. circle, succinate dehydrogenase; , fumarate reductase; -, absorbance at 412 nm.



Prior to ion exchange chromatography, the efficiency of extraction of complex II from mitochondrial membranes was determined with several nonionic detergents. Sucrose monolaurate was the best, considering both the yield and specific activity of the extracted enzyme. Table 1shows that the succinate dehydrogenase activities of both mitochondria increase about 1.5-fold upon extraction. The stimulation of activity of complex II upon detergent extraction has been noted previously(15, 22) . Elution from a DEAE-Cellulofine column further increased the specific activities 13-fold in both cases. This simple and rapid analytical protocol was also effective for the purification of the complex II. The adult complex II appeared to be over 80% homogeneous, as estimated from the pattern on SDS gel electrophoresis. A high content of complex II in the adult mitochondria (about 8%; see (6) ) is one of the major reasons for the high purity. The specific activity of succinate dehydrogenase in the larval complex II was also high, and higher than that of the adult enzyme (Table 1), although the purity of the larval enzyme estimated by SDS gel electrophoresis was less than 50%. This may be because of the higher turnover number (shown in parentheses in Table 2) of the larval complex II than that of the adult enzyme. In contrast to the high succinate dehydrogenase activity, the fumarate reductase activity of larval complex II was extremely low (less than 3% of that of adult enzyme) as shown in Table 1. Only one peak of succinate dehydrogenase activity was found for both the larval and adult complexes, but the salt concentrations of the peak fraction were different. Larval complex II eluted at about 15 mM NaCl, whereas adult complex II eluted at about 32 mM NaCl. These elution profiles were reproducible even during rechromatography and when different preparations of larval and adult mitochondria were examined. These results indicate that larval complex II differs from adult complex II.





Properties of Larval and Adult Complex II

The enzymatic properties of complexes from both stages were compared. K(m) values for the electron acceptor, ubiquinone-1, in the succinate-ubiquinone-1 reductase were almost the same in both complexes (3 µM). These activities of complexes from both the larval and adult were relatively insensitive to the inhibitors of the mammalian complex II. Only 15% of the succinate dehydrogenase activities of nematode complexes were inhibited by 2-thenoyltrifluoroacetone (0.1 mM) and 3-methyl carboxin (1 µM), whereas more than 80% of the activities of bovine enzyme were inhibited by these compounds at the same concentrations. In contrast to these similarities, the K(m) values for succinate and fumarate were significantly different between the two complexes (Table 2). The complex isolated from L2 had a higher affinity for succinate in the succinate dehydrogenase assay than the adult complex, whereas the complex isolated from the adult had a higher affinity for fumarate in the fumarate reductase assay than the L2 complex.

Like the complex II eluted from gel permeation column, complex II of the larvae as well as adult purified by a DEAE-Cellulofine column contained cytochrome. The absorption peak (402 nm) of the larval complex II, which is the Soret band of the oxidized form of cytochrome b subunit was different from that of the adult complex II (410 nm) as shown in Fig. 2, although the redox difference spectrum in the alpha-region could not be obtained because of the limited amount of larval mitochondria. The absorption peak of cytochrome b in adult complex II was slightly different from that reported previously (413 nm in (5) ). This may be because of the different assay conditions, including detergents used in the previous study. Most cytochromes of larval mitochondria except cytochrome b in the complex II were retained in the column, even after washing the column with the buffer containing 0.15 M NaCl.


Figure 2: Absorption spectra of cytochrome b in complex II eluted from DEAE-Cellulofine. Absorption spectra of the airoxidized form of cytochrome b in the partially purified complex II from adult (A) and L2 (B). A, complex II eluted from DEAE-Cellulofine from A. suum adult (38 µg of protein/ml). B, complex II eluted from DEAE-Cellulofine from A. suum L2 (28 µg of protein/ml).



To investigate the subunit composition and antigenic properties of larval complex II, Western blotting analysis was performed as shown in Fig. 3. Antibodies against the adult Fp and Ip subunits recognized the corresponding subunits of the larval complex II, although the migration of larval Fp was slightly faster than that of the adult Fp (Fig. 3A, lane 1). In contrast to these two catalytic subunits, no cross-reaction was observed when antisera to the adult cybS was immunoblotted against larval complex II (Fig. 3B, lane 1). Differences between the Fp subunits were analyzed further by two-dimensional gel electrophoresis. Two spots were observed when a mixture of the two enzymes was analyzed (Fig. 4, C and D). Difference between the two Fp subunits was also confirmed by the patterns of the peptide map obtained with trypsin and V8 protease as shown in Fig. 5. Almost identical patterns between the digests of larval and adult Ip were found when the extracts from both mitochondria were partially digested by V8 protease (data not shown).


Figure 3: Western blotting with antibodies against adult A. suum complex II. A, monoclonal antibody against adult Fp. B, polyclonal antibodies against adult Ip and cybS. Lane 1, L2 complex II eluted from DEAE-Cellulofine (2.0 µg). Lane 2, adult complex II eluted from DEAE-Cellulofine (1.0 µg). Prestained molecular markers (Bio-Rad) were as follows: phosphorylase B (106 kDa), bovine serum albumin (80 kDa), ovalubmin (49.5 kDa), carbonic anhydrase (32.5 kDa), soybean trypsin inhibitor (27.5 kDa), and lysozyme (18.5 kDa).




Figure 4: Two-dimensional gel electrophoresis of Fp. Proteins were separated as described under ``Materials and Methods,'' and the Fps were detected by the antibody. A, adult mitochondria (9 µg). B, L2 mitochondria (21 µg). C and D (magnified C), mixture of adult and L2 mitochondria (9 and 21 µg, respectively). Prestained molecular markers were the same as those used in Fig. 3.




Figure 5: Peptide maps of Fp. Proteolytic digestions and detection of the peptides by the antibody were performed as described under ``Materials and Methods.'' A, digestion with trypsin (1 µg) for 1 h at 37 °C. B, digestion with V8 protease (3 µg) for 3 h at 37 °C. Lane 1, extract from A. suum adult mitochondria (20 µg). Lane 2, extract from L2 mitochondria (40 µg). Peptides were detected by Western blotting using monoclonal antibody against adult Fp.




DISCUSSION

The data presented here clearly demonstrate the stage-specific expression of two different forms of complex II in the A. suum mitochondria. This conclusion is based upon four definitive observations. First, the larval and adult complex II eluted at different salt concentrations during ion exchange column chromatography. Second, both complexes have different enzymatic properties. The fumarate reductase activity of larval complex II was less than 3% that of adult enzyme, whereas succinate dehydrogenase activities of both complexes are comparable. Third, the Fp subunits from both complexes migrate differently during two-dimensional gel electrophoresis and show different patterns in the peptide map. Fourth, Soret peaks of cytochrome b in both complexes were different, and the cybS subunits from both complexes differ in reactivity against anti-adult cybS antibody. The difference in isoelectric point of the Fps may contribute to the difference in elution profiles of two complexes from DEAE-Cellulofine because the larval Fp migrated to a more basic field in Fig. 4. The Fp subunit appears to contain the substrate-binding active site(9, 10) . We have cloned and sequenced the cDNA for the Fp of the adult A. suum complex II (17) and found a unique cysteine residue (Cys-285) in the active site of nematode Fp. Chemical modification of this cysteine residue resulted in a marked decrease in affinity for succinate(17) , indicating that the different kinetic parameters of complex II from L2 and adult may be caused by a difference in the primary structure and the spatial arrangement of the active site in the Fp subunits.

The presence of b cytochrome in complex II as hydrophobic membrane-anchoring peptides, is a general feature in mitochondria and bacteria(9, 10) . Cytochrome b of the adult complex II is reducible by succinate, and its positive midpoint redox potential (E(m)) (-34 mV) (23) compared with that of cytochrome b of bovine heart (-185 mV)(24) facilitates electron transfer to the succinate/fumarate couple (+30 mV) from the low potential quinone, rhodoquinone (-39 mV)(25) , present in adult mitochondria. Based on the elution profiles of larval complex II and those of cytochromes from the columns shown in the present study (Fig. 1B), the larval complex II also appears to contain a b cytochrome. The E(m) value of the cytochrome b in larval complex II seems to be lower than that of adult because the cytochrome b of complex II is not detectable in larval mitochondria after succinate reduction (8) . This result together with the fact that the electron acceptor from cytochrome b, in larval mitochondria is the high potential quinone, ubiquinone (+110 mV)(8) , suggests that the cytochrome b of the larval complex II is likely to be different from that of adult enzyme. The differences of Soret peak in the spectra (Fig. 2) and in reactivity of both complexes against anti-adult cybS antibody (Fig. 3B) are consistent with this idea, although further analysis on cybL will be required.

Thus, A. suum adapts itself to environmental and physiological changes during its life cycle by modulating energy metabolism and its respiratory chain. Complex II from L2 and the adult function as succinate dehydrogenase in the aerobic respiratory chain and fumarate reductase in the anaerobic respiratory chain, respectively. The data in the present report indicate that these activities appear to be catalyzed by two distinct enzyme complexes, and complex II in larval mitochondria is more similar to aerobic mammalian enzymes with low fumarate reductase activity. Among the four subunits in the complex II, at least Fp and the small subunit of cytochrome b (cybS) of larval complex were different from those of adult complex II. Tissue-specific isoforms of complex II were not found in the recent studies on the human Fp(26, 27) , in contrast to tissue-specific and stage-specific isoforms of cytochrome oxidase(28, 29) . The present study is the first direct indication of stage-specific isoforms of mitochondrial complex II.


FOOTNOTES

*
This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas 04266105 and 06454198 from the Ministry of Education, Science, and Culture of Japan and by a grant from the Naito Foundation (to K. K.). This work was also supported by Grant-in-aid 05670240 from the Ministry of Education, Science, and Culture of Japan (to S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183, Japan.

To whom reprint requests and correspondence should be addressed: Dept. of Parasitology, The Inst. of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Tel.: 81-3-5449-5370; Fax: 81-3-5449-5410.

(^1)
The abbreviations used are: Fp, flavoprotein; Ip, iron-sulfur protein; cybL, large subunit of cytochrome b; cybS, small subunit of cytochrome b.


ACKNOWLEDGEMENTS

We thank Dr. Imajoh-Ohmi (The University of Tokyo) for the technical suggestion for two-dimensional gel electrophoresis and Dr. Komuniecki (Toledo University) for critical reading of the manuscript.


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