ISOLATION OF THE ROTENONE-SENSITIVE NADH-UBIQUINONE REDUCTASE (COMPLEX-I) FROM RED BEET MITOCHONDRIA

Complex I of the respiratory chain (EC 1.6.5.3), measured as NADH-duro quinone and NADH-ubiquinone, reductase activities, was isolated from p urified red beetroot (Beta vulgaris L.) mitochondria. The mitochondria were disrupted by freeze-thawing and inner membrane vesicles were pel leted. After solubilization of the vesicles with Triton X-100, the enz yme complex was purified 11-fold (compared to the activity in the inne r membrane vesicles) by size-exclusion chromatography on a Sephacryl S -400 HR column and then by ion-exchange chromatography on a DEAE-Sepha rose CI-6B column. Triton X-100 was present throughout the purificatio n procedure. The 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 to the pattern obta ined with complex I from Neurospora crassa. Analysis by native-SDS 2-d imensional 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 act ivity had a K(m) (NADH) of about 1 muM and was inhibited by both roten one and dicyclohexylcarbodiimide.