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- Rasmusson, A. G., et al.
(författare)
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Isolation of the rotenome-sensitive NADH-ubiquinone reductase (complex I) from red beet mitochondria
- 1994
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 90:3, s. 607-615
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Tidskriftsartikel (refereegranskat)abstract
- Complex 1 of the respirator) chain (EC 1.6.531, measured as NADH-duroquinone and NADH-ubiquinone, reductase activities, was isolated from purified red beetroot (Beta vulgaris L.I mitochondria. The mitochondria were disrupted by freeze-thawing and inner membrane vesicles were pelleted. After solubilization of the vesicles with Triton X-100, the enzyme complex was purified 11-fold (compared to the activity in the inner membrane vesicles) by size-exclusion chromatography on a Sephacryl S-400 HR column and then by ion-exchange chromatography on a DEAE-Sepharose CL-6B column. Triton X-100 was present throughout the purification procedure. Tire purified complex showed approximately 30 bands on SDS-PAGE and about 15 polypeptides including those at 80. 54, 53. 51. 27. 25 and 22 kDa cross-reacted with polyclonal antibodies raised against complex I from Neurospora crassa. This is similar lo the pattern obtained with complex I from Neurospera crassa.Analysis by nativc-SDS 2-dimensional PAGE revealed the existence of several molecular mass forms of the purified complex.After reconstitution of the purified complex into phosphatidylcholine vesicles, the NADH-ubiquinone reductase activity had a Km (NADH) of about I μM and was inhibited by both rotenone and dicyclohexylcarbodiimide.
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