scholarly journals Control of respiration rate in non-growing cells of Paracoccus denitrificans

1987 ◽  
Vol 246 (3) ◽  
pp. 779-782 ◽  
Author(s):  
I Kucera ◽  
L Lampardová ◽  
V Dadák
Keyword(s):  
Respiration Rate ◽  
Membrane Fraction ◽  
Nadh Dehydrogenase ◽  
Paracoccus Denitrificans ◽  
Direct Determination ◽  
Limiting Factor ◽  
Control Of Respiration ◽  
Ubiquinone Oxidoreductase ◽  
Nadh Ubiquinone Oxidoreductase

By means of fluorimetric measurement and by direct determination of intracellular NAD+ and NADH contents, it was proved that the respiration rate of Paracoccus denitrificans cells utilizing glucose is limited by processes preceding NADH oxidation in the respiratory chain, so that the membrane NADH dehydrogenase is not saturated by its substrate. In the separated membrane fraction on saturation with exogenous NADH the main limiting factor is represented by NADH: ubiquinone oxidoreductase.

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 Photoaffinity labelling of mitochondrial NADH dehydrogenase with arylazidoamorphigenin, an analogue of rotenone

1984 ◽  
Vol 224 (2) ◽  
pp. 525-534 ◽  
Author(s):  
F G P Earley ◽  
C I Ragan
Keyword(s):  
Nadh Dehydrogenase ◽  
Oxidoreductase Activity ◽  
Photoaffinity Labelling ◽  
Ubiquinone Oxidoreductase ◽  
Naturally Occurring ◽  
Respiratory Inhibitor ◽  
Nadh Ubiquinone Oxidoreductase ◽  
Low Concentrations ◽  
Preferential Incorporation ◽  
Parallel Fashion

A photoaffinity-labelling analogue of the respiratory inhibitor rotenone was synthesized from the naturally occurring rotenoid amorphigenin. The analogue inhibits NADH-ubiquinone oxidoreductase activity at concentrations comparable with those of rotenone. Photolysis of the radiolabelled analogue bound to isolated NADH-ubiquinone oxidoreductase resulted in preferential incorporation of radioactivity into a polypeptide of Mr 33 000, particularly at low concentrations of the inhibitor. Preparations of the enzyme differ in a parallel fashion in the content of this polypeptide, the degree of photolabelling by the analogue and their sensitivity to rotenone, providing further evidence that the 33 000-Mr protein forms part of the rotenone-binding site.

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