Plasma membrane 6-cytochromes

All these intermediates except for cytochrome c are membrane-associated (either in the mitochondrial inner membrane of eukaryotes or in the plasma membrane of prokaryotes). All three types of proteins involved in this chain— flavoproteins, cytochromes, and iron-sulfur proteins—possess electron-transferring prosthetic groups. 

The electron transport chain system responsible for the respiratory burst (named NADPH oxidase) is composed of several components. One is cytochrome 6558, located in the plasma membrane it is a heterodimer, containing two polypeptides of 91 kDa and... 

Figure 12.2 Copper chaperone function, (a) Copper homeostasis in Enterococcus hirae is affected by the proteins encoded by the cop operon. CopA, Cu1+-import ATPase CopB, Cu1+-export ATPase CopY, Cu1+-responsive repressor copZ, chaperone for Cu1+ delivery to CopY. (b) The CTR family of proteins transports copper into yeast cells. Atxlp delivers copper to the CPx-type ATPases located in the post Golgi apparatus for the maturation of Fet3p. (c) Coxl7p delivers copper to the mitochondrial intermembrane space for incorporation into cytochrome c oxidase (CCO). (d) hCTR, a human homologue of CTR, mediates copper-ion uptake into human cells. CCS delivers copper to cytoplasmic Cu/Zn superoxide dismutase (SOD1). Abbreviations IMM, inner mitochondrial membrane OMM, outer mitochondrial membrane PM, plasma membrane PGV, post Golgi vessel. Reprinted from Harrison et al., 2000. Copyright (2000), with permission from Elsevier Science.

A further promising attempt to identify the bluelight-reducible cyt b is described by Britz et al.26) for a plasma-membrane-enriched fraction of com coleoptiles. They find that methylene blue (Scheme 2) is capable of reducing a particular cyt b which constitutes only 10—20% of the total dithionite-reducible cytochromes. Since this particular cyt b is very similar to that which is photo-reduced by endogeneous flavin in Neurospora 122,123) the two are proposed to be identical. 

Robin, M.A. et al., Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes, J. Pharmacol. Exp. Ther., 294, 1063, 2000. 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

Figure 5.7. Translocation of cytochrome b to the plasma membrane. In non-stimulated cells, only a small proportion of the total cellular pool of cytochrome b is present on the plasma membrane. The major pool of this cytochrome is located on the membranes of specific granules, gelatinase-containing granules and secretory vesicles. During activation (e.g. by fMet-Leu-Phe or PMA) or priming (e.g. by cytokines), some of these subcellular pools of cytochrome b molecules are translocated to the plasma membrane, thereby increasing the level of surface cytochrome b.

Experiments in the mid-1980s, analysing the phosphorylation profiles of neutrophils from autosomal recessive forms of CGD, indicated that a component of around 45-47 kDa failed to become phosphorylated during activation with PMA. In normal neutrophils, this component is phosphorylated in the cytoplasm and then translocates to the plasma membrane. Curiously, in CGD neutrophils of patients that lacked the cytochrome b, the 47-kDa component was phosphorylated normally but failed to become incorporated into the plasma membrane. This strongly implied the following ... 

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. 

Cross, A. R., Harper, A. M., Segal, A. W. (1981). Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. Biochem. J. 194, 599-606. 

It is known that protein kinase C can phosphorylate a number of key oxidase components, such as the two cytochrome b subunits and the 47-kDa cytoplasmic factor. This process is prevented by protein kinase C inhibitors such as staurosporine (although it is now recognised that this inhibitor is not specific for protein kinase C), which also inhibits the respiratory burst activated by agonists such as PMA. However, when cells are stimulated by fMet-Leu-Phe, translocation of pAl-phox to the plasma membrane can occur even if protein kinase C activity is blocked - that is, phosphorylation is not essential for the translocation of this component in response to stimulation by this agonist. Similarly, the kinetics of phosphorylation of the cytochrome subunits do not follow the kinetics of oxidase activation, and protein kinase C inhibitors have no effect on oxidase activity elicited by some agonists -for example, on the initiation of the respiratory burst elicited by agonists such as fMet-Leu-Phe (Fig. 6.14). Furthermore, the kinetics of DAG accumulation do not always follow those of oxidase activity. Hence, whilst protein kinase C is undoubtedly involved in oxidase activation by some agonists, oxidase function is not totally dependent upon the activity of this kinase. 

The second major breakthrough in understanding the defect in CGD neutrophils came through the development of assays in which the NADPH oxidase can be activated in a cell-free system in vitro ( 5.3.2.3). In these systems, activation of the oxidase can be achieved by the addition of cytoplasm to plasma membranes in the presence of NADPH and arachidonic acid (or SDS or related substances). Interestingly, the oxidase cannot be activated in these cell-free systems using extracts from CGD neutrophils however, cytosol and plasma membranes from normal and CGD neutrophils may be mixed, and in most cases activity is restored if the correct mixing pattern is used. For example, as may be predicted, in X-linked CGD it is the membranes that are defective (because the cytochrome b is deficient), whereas in autosomal recessive CGD the cytosol is defective in the cell-free system. 

Kawai, K., Tyurina, Y.Y, Tyurin, V.A., Kagan, V.E., and Fabisiak, J., 2000, Peroxidation and externalization of phosphatidylserine in plasma membrane of HL-60 ceUs during tert-butyl hydroperoxide-induced apoptosis Role of cytochrome c, The Toxicologist 54 SuppL 776. 

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