Molecular & Biochemical Parasitology 128 (2003) 175–186 Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria Hisako Amino a , Arihiro Osanai a , Hiroko Miyadera a , Noriko Shinjyo a , Eriko Tomitsuka a , Hikari Taka b , Reiko Mineki b , Kimie Murayama b , Shinzaburo Takamiya c , Takashi Aoki c , Hideto Miyoshi d , Kimitoshi Sakamoto d , Somei Kojima e , Kiyoshi Kita a,∗ a Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan b Division of Biochemical Analysis, Central Laboratory of Medical Sciences, School of Medicine, Juntendo University, Tokyo, Japan c Department of Parasitology, School of Medicine, Juntendo University, Tokyo, Japan d Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan e Department of Parasitology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Received 10 December 2002; received in revised form 11 March 2003; accepted 11 March 2003 Abstract We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron–sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS L ) is distinct from the adult CybS (CybS A ). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS L and CybS A in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K m values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Ascaris suum; Succinate dehydrogenase; Fumarate reductase; Large subunit of cytochrome b; Small subunit of cytochrome b Abbreviations: Fp, flavoprotein; Ip, iron–sulfur cluster subunit; L3, third stage larva; CybL, large subunit of cytochrome b; CybS, small subunit of cytochrome b; Fp L , larval Fp, Fp A ; adult Fp; CybS L , larval CybS, CybS A ; adult CybS; PCR, polymerase chain reaction; RT-PCR, re- verse transcription PCR; SDH, succinate dehydrogenase; FRD, fumarate reductase; SQR, succinate-ubiquinone reductase; QFR, quinol-fumarate reductase; RQFR, rhodoquinol-fumarate reductase; SL, spliced leader; RACE, rapid amplification of cDNA ends; Dig, Digoxigenin; NJ, neigh- bor joining; ML, maximum likelihood; SH, Shimodaira–Hasegawa; PGL, paraganglioma; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; CBB, Coomassie brilliant blue; HPLC, high performance liquid chromatography ∗ Corresponding author. Tel.: +81-3-5841-3526; fax: +81-3-5841-3444. E-mail address: kitak@m.u-tokyo.ac.jp (K. Kita). 1. Introduction The mitochondrial respiratory chain of the parasitic ne- matode, Ascaris suum, changes dramatically during its life cycle [1–7]. A. suum larvae in the free-living stage inhabit the air (∼160 mmHg), whose mitochondria utilize clas- sic mammalian-type aerobic respiration [1,2]. In contrast, the mitochondria of the adult worm utilize an anaerobic NADH-fumarate reductase system as a major component of the respiratory chain [2]. This switch allows respiration by the adult worm in the host small intestine where there is limited oxygen tension (0–10 mmHg) [2]. Quinones, low molecular weight electron carriers that transfer electrons between respiratory complexes, also change markedly dur- ing the A. suum life cycle. The larvae employ ubiquinone 0166-6851/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0166-6851(03)00074-4