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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... (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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Please use this url to cite or link to this publication:
author
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
Wiley-Blackwell
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
2016-10-23 04:43:38
@misc{3407dfee-7c7d-4c09-8159-4dcc30069206,
  abstract     = {<br/>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},
  keyword      = {Beta vulgaris,complex I,inner membrane,mitochondrion,NADH dehydrogenase,red beetroot},
  language     = {eng},
  number       = {3},
  pages        = {607--615},
  publisher    = {ARRAY(0x8355f18)},
  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},
  volume       = {90},
  year         = {1994},
}