Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line.
Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.
Schilling
Birgit
B
Buck Institute for Age Research, Novato, CA 94945, USA.
Bharath M M
Srinivas
S
Row
Richard H
RH
Murray
James
J
Cusack
Michael P
MP
Capaldi
Roderick A
RA
Freed
Curt R
CR
Prasad
Kedar N
KN
Andersen
Julie K
JK
Gibson
Bradford W
BW
D000818:Animals; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D009474:Neurons / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D021122:Protein Subunits; D042967:Electron Transport Complex I; D003545:Cysteine; D004298:Dopamine