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Isolation of the rotenome-sensitive NADH-ubiquinone reductase (complex I) from red beet mitochondria

Rasmusson, A. G. LU ; Mendel-Hartvig, J. ; Moller, Ian M. and Wiskich, J. T. (1994) In Physiologia Plantarum 90(3). p.607-615
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... (More)
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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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Beta vulgaris, complex I, inner membrane, mitochondrion, NADH dehydrogenase, red beetroot
in
Physiologia Plantarum
volume
90
issue
3
pages
607 - 615
publisher
John Wiley & Sons Inc.
external identifiers
  • scopus:0028025890
ISSN
0031-9317
DOI
10.1034/j.1399-3054.1994.900324.x
language
English
LU publication?
yes
id
3407dfee-7c7d-4c09-8159-4dcc30069206
date added to LUP
2016-05-31 21:37:40
date last changed
2022-04-11 14:06:54
@article{3407dfee-7c7d-4c09-8159-4dcc30069206,
  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.<br/>Analysis by nativc-SDS 2-dimensional PAGE revealed the existence of several molecular mass forms of the purified complex.<br/>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.<br/>}},
  author       = {{Rasmusson, A. G. and Mendel-Hartvig, J. and Moller, Ian M. and Wiskich, J. T.}},
  issn         = {{0031-9317}},
  keywords     = {{Beta vulgaris; complex I; inner membrane; mitochondrion; NADH dehydrogenase; red beetroot}},
  language     = {{eng}},
  number       = {{3}},
  pages        = {{607--615}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{Physiologia Plantarum}},
  title        = {{Isolation of the rotenome-sensitive NADH-ubiquinone reductase (complex I) from red beet mitochondria}},
  url          = {{http://dx.doi.org/10.1034/j.1399-3054.1994.900324.x}},
  doi          = {{10.1034/j.1399-3054.1994.900324.x}},
  volume       = {{90}},
  year         = {{1994}},
}