Components of the oxidase

Over the years, there have been numerous reports of oxidase preparations that contain polypeptide components, additional to those described above. As yet no molecular probes are available for these, and so their true association with the oxidase is unconfirmed. There are many reports in the literature describing the role of ubiquinone as an electron transfer component of the oxidase, but its involvement is controversial. Quinones (ubiquinone-10) have reportedly been detected in some neutrophil membrane preparations, but other reports have shown that neither plasma membranes, specific granules nor most oxidase preparations contain appreciable amounts of quinone, although some is found in either tertiary granules or mitochondria. Still other reports suggest that ubiquinone, flavoprotein and cytochrome b are present in active oxidase preparations. Thus, the role of ubiquinone and other quinones in oxidase activity is in doubt, but the available evidence weighs against their involvement. Indeed, the refinement of the cell-free activation system described above obviates the requirement for any other redox carriers for oxidase function. 

Messenger RNA molecules for both subunits of the cytochrome and the two cytosolic components are detectable in unstimulated bloodstream cells. Experiments involving incubation of neutrophil suspensions with the protein synthesis inhibitor cycloheximide indicate that constitutive expression of one or more components of the oxidase is required for the neutrophil to maintain its ability to generate reactive oxidants. For example, when neutrophils are incubated in vitro with cycloheximide, their ability to generate reactive oxidants declines more rapidly than in control cells, as they age in culture (Fig. 7.12). This decline in oxidase activity when protein biosynthesis is blocked is not due to cell death, because cells treated with cycloheximide for this time still exclude trypan blue. Furthermore, when protein biosynthesis is stimulated in neutrophils by the addition of GM-CSF for 24 h in vitro, the ability to generate reactive oxidants is enhanced considerably above the levels observed in untreated cells. 

Purification of p-hydroxyphenylpyruvic oxidase led to the separation of two proteins, neither of which is active alone. One of these was identified as catalase. Catalase can be replaced as a component of the oxidase system by a peroxidase. These findings implicate H2O2 in the oxidation, and this is supported by the elimination of a lag period in the reaction by the addition of small quantities of H2O2. In spite of the requirement for two proteins and the effect of H2O2, an intermediate-level oxidant, no... 

Molybdenum. Molybdenum is a component of the metaHoen2ymes xanthine oxidase, aldehyde oxidase, and sulfite oxidase in mammals (130). Two other molybdenum metaHoen2ymes present in nitrifying bacteria have been characteri2ed nitrogenase and nitrate reductase (131). The molybdenum in the oxidases, is involved in redox reactions. The heme iron in sulfite oxidase also is involved in electron transfer (132). 

NADPH-oxidase j 2O2 + NADPH 20p + NADP -F H+ Key component of the respiratory burst Deficient in chronic granulomatous disease... 

Mutations in the Genes for Components of the NADPH Oxidase System Cause Chronic Granulomatous Disease... 

As noted, the alkaloid yield from the Beocin plants was low, which the authors suggested might be caused by the poor soil in which the plants were growing (Popovic et ah, 1992). One could ask whether the soil conditions to which they refer might be influential in the overall alkaloid biosynthetic processes in this species. It would be of interest to see experimental studies aimed at determining the effect of soil components on these processes. In the present case, it may be a lack of, or reduction in the activity of, the oxidase(s) necessary for the dimerization process (required to form the bibenzyldihydroisoquinolines) to occur. It is also possible that the lack of dimeric alkaloids may simply reflect a concentration effect caused by the edaphic conditions. These questions should be accessible to experiment. 

P. Mitchell (Nobel Prize for Chemistry, 1978) explained these facts by his chemiosmotic theory. This theory is based on the ordering of successive oxidation processes into reaction sequences called loops. Each loop consists of two basic processes, one of which is oriented in the direction away from the matrix surface of the internal membrane into the intracristal space and connected with the transfer of electrons together with protons. The second process is oriented in the opposite direction and is connected with the transfer of electrons alone. Figure 6.27 depicts the first Mitchell loop, whose first step involves reduction of NAD+ (the oxidized form of nicotinamide adenosine dinucleotide) by the carbonaceous substrate, SH2. In this process, two electrons and two protons are transferred from the matrix space. The protons are accumulated in the intracristal space, while electrons are transferred in the opposite direction by the reduction of the oxidized form of the Fe-S protein. This reduces a further component of the electron transport chain on the matrix side of the membrane and the process is repeated. The final process is the reduction of molecular oxygen with the reduced form of cytochrome oxidase. It would appear that this reaction sequence includes not only loops but also a proton pump, i.e. an enzymatic system that can employ the energy of the redox step in the electron transfer chain for translocation of protons from the matrix space into the intracristal space. 

Knoller, S., Shpungin, S., and Pick, E. (1991) The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559./. Biol. Chem. 266, 2795-2804. 

Lipid-soluble xenobiotics are commonly biotra ns formed by oxidation in the drug-metabolizing microsomal system (DMMS). For each description below, choose the component of the microsomal mixed-function oxidase system with which it is most closely associated ... 

The NADPH oxidase is in fact a multicomponent enzyme system that constitutes an electron transport chain from NADPH to O2. The components of this oxidase complex are now almost completely defined, and experiments performed primarily with CGD neutrophils have helped to identify these major constituents. 

In the early 1960s in Japan, a b-type cytochrome was found in horse neutrophils and, because it bound CO, it was proposed to be functional during the respiratory burst. This work went largely unnoticed, but in 1978 Segal and Jones in the United Kingdom discovered that a b-type cytochrome became incorporated into phagolysosomes furthermore, this cytochrome was absent in some patients with CGD. These workers correctly proposed that it was a key component of the NADPH oxidase. This cytochrome was a landmark discovery in phagocyte research for a number of reasons ... 

At around the same time as these discoveries, several groups were trying to determine the ways in which the NADPH oxidase could be activated in vitro (i.e. activated in broken-cell suspensions). The ultimate aim of such studies was to determine the minimal components necessary for assembly and activation of the oxidase in in vivo experiments. Thus, once these minimal constituents were identified, oxidase activity could then be reconstituted in vitro from the individual component parts. The first breakthrough in these studies was... 

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