ABSTRACT
We evaluated electron transport chain activity in platelet mitochondria taken from HD patients. All 5 patients studied had striking depressions of NADH:ubiquinone oxidoreductase activity (complex I) (5.36 +/- 2.91 nmol/min/mg; control mean, 19.12 +/- 5.64 nmol/min/mg). Other electron transport chain activities were not significantly different from control values. HD may be caused by a mutation in 1 of the nuclear coded subunits of NADH:ubiquinone oxidoreductase.
Subject(s)
Blood Platelets/enzymology , Huntington Disease/enzymology , Quinone Reductases/blood , Electron Transport , Female , Humans , Huntington Disease/blood , Huntington Disease/genetics , Male , Middle Aged , Mitochondria/enzymology , NAD(P)H Dehydrogenase (Quinone) , Quinone Reductases/deficiency , Reference Values , Risk FactorsABSTRACT
The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.
Subject(s)
NADH, NADPH Oxidoreductases/blood , Neutrophils/enzymology , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Cytochrome b Group/blood , Flavin-Adenine Dinucleotide/blood , NADH, NADPH Oxidoreductases/isolation & purification , NADPH Oxidases , NADPH-Ferrihemoprotein Reductase/blood , Quinone Reductases/blood , Subcellular Fractions/enzymology , Swine , Ubiquinone/bloodABSTRACT
Superoxide (O-2) production by partially purified NADPH oxidase from guinea pig neutrophils was markedly increased when the cells were activated by exposure to phorbol-myristate acetate. On the contrary, NADPH-dependent cytochrome c and 2,6-dichlorophenolindophenol (DCIP) reductase activities in preparations from resting and activated neutrophils were similar. The apparent Km values for NADH and NADPH of the reductase activities were different from those of the O-2 producing enzyme. The electron acceptors did not inhibit the oxygen consumption by NADPH oxidase in the presence of superoxide dismutase. Even in anaerobiosis the oxidase failed to reduce cytochrome c and DCIP. These results suggest that NAD(P)H-dependent dye reductase activities are not involved in the electron transport system responsible for the O-2 production by neutrophils.
Subject(s)
2,6-Dichloroindophenol/metabolism , Cytochrome c Group/metabolism , Indophenol/analogs & derivatives , NADH, NADPH Oxidoreductases/blood , Neutrophils/enzymology , Superoxides/metabolism , Anaerobiosis , Animals , Guinea Pigs , Kinetics , NADPH Oxidases , NADPH-Ferrihemoprotein Reductase/blood , Oxygen Consumption , Quinone Reductases/blood , Xanthine Oxidase/metabolismABSTRACT
NADPH-dependent ubiquinone-1 reductase activity was present in the phagocytic vesicles of pig polymorphonuclear leucocytes. The apparent Km-value of the reductase for NADPH was 29 microM which is similar to that of the NADPH-dependent superoxide formation. Increase of the quinone-reductase activity by increasing the concentrations of ubiquinone-1 was associated with the decrease of the superoxide forming activity, the rate of the NADPH oxidation being constant independent of the quinone concentration. p-Chloromercuribenzoate inhibited both superoxide formation and reduction of the quinone, whereas low concentrations of cetyltrimethylammonium bromide which inhibit the superoxide formation did not inhibit the reduction of the quinone. The reduction of 2,6-dichlorophenolindophenol which has been shown not to be inhibited by both inhibitors. The quinone-reductase activity could be extracted with a mixture of deoxycholate and Tween 20 which extracts the superoxide forming activity. The observations indicate that a region of the superoxide-forming NADPH oxidase between a mercurial-sensitive site and a site sensitive to the cationic detergent is responsible for the reduction of ubiquinone.
Subject(s)
NADH, NADPH Oxidoreductases/blood , NADPH Oxidases , Neutrophils/enzymology , Quinone Reductases/blood , Animals , Electron Transport , Kinetics , NAD(P)H Dehydrogenase (Quinone) , Neutrophils/physiology , Phagocytosis , SwineABSTRACT
Four catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils have been identified. DCIP reductase, cytochrome c reductase and a chromophore 450-455 reductase are present in phorbol myristate acetate stimulated neutrophils and absent in resting cells and phorbol myristate acetate stimulated chronic granulomatous disease cells. Quinol dehydrogenase activity has also been demonstrated in activated and resting cells. Furthermore, a chromophore absorbing in the reduced state at 450-455 nm participates in superoxide production. This chromophore is reduced by NADPH or duroquinol and is missing in cell lysates derived from a patient with chronic granulomatous disease.
