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

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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.

Detaljer

Författare
Enheter & grupper
Externa organisationer
  • University of Oregon
  • University of Adelaide
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Naturvetenskap

Nyckelord

Originalspråkengelska
Sidor (från-till)607-615
TidskriftPhysiologia Plantarum
Volym90
Utgåva nummer3
StatusPublished - 1994
PublikationskategoriForskning
Peer review utfördJa