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