Comparative study and cDNA cloning of the flavoprotein subunit of mitochondrial complex II (succinate-ubiquinone oxidoreductase: fumarate reductase) from the dog heartworm, Dirofilaria immitis.
Mitochondrial complex II functions as a fumarate reductase (FRD), the reverse reaction of succinate dehydrogenase (SDH), and plays an important role in the anaerobic respiratory chain of parasitic helminths. In this study, complex II from the dog heartworm, Dirofilaria immitis adult, which is thought to act as a homolactatic fermenter, was examined in terms of its enzymatic features and primary structure in order to investigate the possible role of mitochondria in this filaria. Mitochondria from D. immitis adult showed high FRD activity when the enzymatic assay was performed using methylviologen as an artificial electron donor. The ratio of SDH to FRD in D. immitis was comparable to that in Ascaris suum adult, which is known to have an anaerobic mitochondrial respiratory chain with a high FRD activity of complex II. The FRD activity of D. immitis mitochondria was inhibited by the sulfhydryl reagent N-ethylmaleimide (NEM), while that of A. suum complex II was resistant to this inhibitor. The presence of the flavoprotein (Fp) subunit, which contains the substrate binding active site, was confirmed in D. immitis mitochondria by immunoblotting using a monoclonal antibody against the A. suum Fp subunit. By homology probing with the polymerase chain reaction, the entire cDNA for the D. immitis adult Fp was cloned and sequenced. The deduced amino acid sequence showed significant homology to that of A. suum and other mitochondrial Fps, in contrast to much less similarity to bacterial FRD, even though the D. immitis complex II showed high FRD activity. These results are the first indication of the presence of a functional complex II in D. immitis mitochondria.
Kuramochi
T
T
Department of Veterinary Internal Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan.
Kita
K
K
Takamiya
S
S
Kojima
S
S
Hayasaki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004183:Dirofilaria immitis / Q000201:enzymology* / Q000235:genetics*; D004285:Dogs; D042963:Electron Transport Complex II; D004734:Energy Metabolism; D005260:Female; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics*; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000235:genetics*
D018076:DNA, Complementary; D005420:Flavoproteins; D009097:Multienzyme Complexes; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D016660:NAD(P)H Dehydrogenase (Quinone)