scholarly journals Identification of the subunits of bovine heart mitochondrial NADH dehydrogenase that are exposed to the phospholipid bilayer by photo-labelling with 5-iodonaphth-1-yl azide

1980 ◽  
Vol 191 (2) ◽  
pp. 429-436 ◽  
Author(s):  
F G P Earley ◽  
C I Ragan
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Phospholipid Bilayer ◽  
Hydrophobic Region ◽  
Protein Fragments ◽  
Iron Protein ◽  
Bovine Heart ◽  
Nadh Ubiquinone Oxidoreductase ◽  
Lipoprotein Complex

Mitochondrial NADH dehydrogenase may be isolated from bovine heart as a lipoprotein complex (Complex I or NADH-ubiquinone oxidoreductase). Polypeptide subunits that are exposed to the hydrophobic region of the phospholipid bilayer were identified by photolabelling with the hydrophobic probe, 5-[125I]iodonaphth-1-yl azide. Chaotropic resolution of the labelled enzyme showed that the hydrophilic flavoprotein and iron-protein fragments of the enzyme were not in contact with the phospholipid bilayer. When complex I that had been partially depleted of phospholipids was photolabelled, incorporation of radioactivity into certain polypeptides was increased, indicating either conformational changes in protein or preferential association of these polypeptides with residual cardiolipin. A model NADH dehydrogenase structure is proposed on the basis of these results and those obtained with hydrophilic probes by Smith & Ragan (1980) Biochem. J. 185, 315-326.

scholarly journals Immunochemical probing of the structure and cofactor of NADH dehydrogenase from Paracoccus denitrificans

1987 ◽  
Vol 244 (3) ◽  
pp. 661-668 ◽  
Author(s):  
C L George ◽  
S J Ferguson
Keyword(s):  
Succinate Dehydrogenase ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Plasma Membranes ◽  
Paracoccus Denitrificans ◽  
Ubiquinone Oxidoreductase ◽  
Crossed Immunoelectrophoresis ◽  
Nadh Ubiquinone Oxidoreductase ◽  
Wide Range ◽  
A Subunit

Monospecific antibody to the respiratory NADH dehydrogenase from Paracoccus denitrificans was prepared by using as antigen specific immunoprecipitates containing NADH dehydrogenase which were excised from crossed-immunoelectrophoresis plates. The latter were run with selectively solubilized plasma membranes and antibodies against plasma membranes. The antibody immunoprecipitated NADH dehydrogenase from P. denitrificans membranes biosynthetically labelled with 14C and solubilized with a wide range of detergents. All immunoprecipitates contained the two subunits of Mr 48,000 and 25,000, in an approximate 1:1 stoichiometry, that had previously been assigned to NADH dehydrogenase. A polypeptide of Mr 46,000 in P. denitrificans membranes, previously shown to cross-react with a subunit-specific antibody to mitochondrial NADH dehydrogenase (complex I), was not detected in any immunoprecipitate. Under some conditions a third polypeptide, of Mr 31,000, was also detected, but in variable and non-stoichiometric amounts relative to the two other subunits. It was concluded that this polypeptide was incorporated into the immunoprecipitates as an artefact and that the polypeptides of Mr 48,000 and 25,000 are the sole polypeptides firmly identified in the NADH dehydrogenase. Flavoproteins were specifically radiolabelled by growth of P. denitrificans in the presence of [14C]riboflavin. Crossed immunoelectrophoresis of membranes from such cells showed that succinate dehydrogenase contained flavin, but that there was no detectable flavin in NADH dehydrogenase under these conditions. Analysis of excised immunoprecipitates of succinate dehydrogenase showed that flavin was covalently bound to a polypeptide of Mr 56,000. Flavin was retained by NADH dehydrogenase under mild conditions of detergent solubilization. Subsequent immunoprecipitation, followed by analysis of the acid-extracted flavin, established that FMN is a cofactor, in common with mitochondrial NADH-ubiquinone oxidoreductase (complex I).

scholarly journals An analysis of the polypeptide composition of bovine heart mitochondrial NADH-ubiquinone oxidoreductase by two-dimensional polyacrylamide-gel electrophoresis

1979 ◽  
Vol 181 (2) ◽  
pp. 435-443 ◽  
Author(s):  
C Heron ◽  
S Smith ◽  
C I Ragan
Keyword(s):  
Gel Electrophoresis ◽  
Complex I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Submitochondrial Particles ◽  
Ubiquinone Oxidoreductase ◽  
Bovine Heart ◽  
Nadh Ubiquinone Oxidoreductase

Purified preparations of Complex I (NADH-ubiquinone oxidoreductase) from bovine heart mitochondria may be resolved into 26 polypeptides by two-dimensional analysis combining isoelectric focusing and polyacrylamide-gel electrophoresis in sodium dodecyl sulphate. Similar analyses of the fragments obtained from chaotropic resolution of the enzyme show that each of these fragments contains a distinct and non-overlapping set of polypeptides. Evidence that the polypeptides seen in the intact enzyme are true constituents comes from analyses of immunoprecipitates obtained by allowing Complex I or solubilized submitochondrial particles to react with antisera directed against the whole enzyme and a subfragment of the enzyme.

scholarly journals 3P123 Stabilization of bovine heart NADH-ubiquinone oxidoreductase(complex I) with phospholipids

2005 ◽  
Vol 45 (supplement) ◽  
pp. S234
Author(s):  
H. Sugiyama ◽  
R. Nakatubo ◽  
T. Terada ◽  
K. Itou-Shinzawa ◽  
S. Yoshikawa
Keyword(s):  
Complex I ◽  
Ubiquinone Oxidoreductase ◽  
Bovine Heart ◽  
Nadh Ubiquinone Oxidoreductase

scholarly journals Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart

1985 ◽  
Vol 227 (2) ◽  
pp. 467-474 ◽  
Author(s):  
M W J Cleeter ◽  
S H Banister ◽  
C I Ragan
Keyword(s):  
Ethylene Glycol ◽  
Active Site ◽  
Protein Fraction ◽  
Nadh Dehydrogenase ◽  
Cross Linking ◽  
Iron Protein ◽  
Bovine Heart ◽  
Cross Links ◽  
Hydrophobic Shell ◽  
Chemical Cross Linking

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions.

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