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Isolation of the rotenone‐sensitive NADH‐ubiquinone reductase (complex I) from red beet mitochondria
Author(s) -
Rasmusson Allan G.,
MendelHartvig Janet,
Møller Jan M.,
Wiskich Joseph T.
Publication year - 1994
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1994.tb08821.x
Subject(s) - neurospora crassa , biochemistry , rotenone , vesicle , biology , molecular mass , antimycin a , mitochondrion , chromatography , chemistry , enzyme , membrane , gene , mutant
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 K m (NADH) of about I μ M and was inhibited by both rotenone and dicyclohexylcarbodiimide.