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PLANT PHYSIOLOGY , Vol 102, Issue 2 435-443, Copyright © 1993 by American Society of Plant Biologists
Purification and Preliminary Characterization of Mitochondrial Complex I (NADH:Ubiquinone Reductase) from Broad Bean (Vicia faba L.)
S. Leterme and M. Boutry
Unite de Biochimie Physiologique, Universite Catholique de Louvain, Place Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium
NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from
broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential
treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)
dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH
dehydrogenase activities. Complex I was solubilized in the presence of 4%
Triton X-100 and then purified by sucrose-gradient centrifugation in the
presence of the same detergent. The second purification step was
hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100
helped remove contaminants such as ATPase. The high molecular mass complex
is composed of at least 26 subunits with molecular masses ranging from 6000
to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone
analogs but not cytochrome c. NADPH could not substitute for NADH as an
electron donor. The KM for NADH was 20 [mu]M at the optimum pH of 8.0. The
NH2-terminal sequence of several subunits was determined, revealing the
ambiguous nature of the 42-kD subunit.
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