Purification of the NADH:ubiquinone oxidoreductase (complex I) of the respiratory chain from the inner mitochondrial membrane of Solanum tuberosum.
The plant NADH:ubiquinone oxidoreductase (or complex I) was isolated from potato (Solanum tuberosum) mitochondria. The multisubunit enzyme was solubilized with detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), out of the inner mitochondrial membranes and purified by hydroxylapatite and gel filtration chromatography. The preparation was found to be virtually free of any ATPase or transhydrogenase contamination. Complex I of potato is composed of at least 32 individual subunits as detected in silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a total molecular mass of about 900 kDa. The enzyme preparation showed an NADH:ubiquinone-2 reductase activity of 11.5 mumol x min-1 x mg-1 and is strongly inhibited by rotenone. Heterologous polyclonal antibodies against the 70- and 49-kDa subunits of the Neurospora crassa complex I and against the wheat NAD9 subunit cross-reacted specifically with the respective potato subunits. Four of the 10 NH2-terminal sequences determined show significant similarities to Neurospora or bovine complex I subunits and allow a tentative assignment of these subunits.
Herz
U
U
Institut für Genbiologische Forschung Berlin, Federal Republic of Germany.
Schröder
W
W
Liddell
A
A
Leaver
C J
CJ
Brennicke
A
A
Grohmann
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002850:Chromatography, Gel; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009491:Neurospora / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology*
D046911:Macromolecular Substances; D017886:Durapatite; D016660:NAD(P)H Dehydrogenase (Quinone)