Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils.

A cytochrome b with a midpoint oxidation-reduction potential of -245mV (cytochrome b-245) that is a major component of the microbicidal oxidase system of human neutrophil leucocytes has been identified in human eosinophils, monocytes and macrophages at concentrations similar to that found in human neutrophils. It was absent from a variety of other cells. This cytochrome is present in phagocytic leucocytes and probably plays an important part in the specialized activities of these cells.     

Purification of cytochrome b-245 from human neutrophils.

The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.     

Presence of cytochrome b-245 in NADPH oxidase preparations from human neutrophils

The composition of NADPH oxidase purified by Red Sepharose chromatography of extracts from human neutrophil membranes was investigated. In contrast to that was recently reported by others, the enzyme isolated according to this procedure contained a high concentration of cytochrome b-245 and little FAD. The results reinforce the belief that cytochrome b-245 is a major component of the NADPH oxidase and plays a fundamental role in the formation of O2-by neutrophils.     

Changes in the subcellular distribution of the cytochrome b-245 on stimulation of human neutrophils.

Cytochrome b-245 of neutrophils has a bimodal distribution in sucrose density gradients. The lighter component (d = 1.14) is shown to be associated with the plasma membrane by the similarity between its density and that of markers of this organelle, as well as a parallel increase in the density of the cytochrome and plasma membrane after treatment with digitonin or dimethyl suberimidate. The cytochrome b-245 of monocytes and cytoplasts, the latter produced by the removal of nuclei and granules from neutrophils, was located only in the plasma membrane. The denser peak of cytochrome (d = 1.19), which contained approximately half of the cytochrome b of neutrophils, had a similar density-distribution profile to the specific granules. After hypo-osmotic disruption of this denser material, the cytochrome distributed with the density of membranes, suggesting an original location within the membrane of the intracellular structure. Redistribution of the cytochrome from the granules to the membranes was observed after stimulation of respiratory activity with soluble agents or opsonized particles. This translocation is not responsible for activation of the oxidase system. There was poor agreement between the kinetics of the transfer of cytochromes from the dense component to the membranes, and degranulation of specific-granule contents, suggesting that the cytochrome may be located in another intracellular structure or that its localization becomes further modified after granule fusion.     

The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.

1. The absorption coefficient of human neutrophil plasma-membrane reduced-minus-oxidized cytochrome b-245 was determined [delta epsilon (mM; 559-540 nm) = 21.6 cm-1]. 2. Neutrophil polymorphonuclear leucocytes (neutrophils) were prepared from human, ox, horse and pig blood. In each case plasma-membrane fractions were found to contain low-potential cytochrome b. When membranes from horse neutrophils were incubated anaerobically with either NADH or NADPH the cytochrome b became reduced. Prior stimulation of the cells with phorbol myristate acetate did not increase the rate or extent of cytochrome b reduction in isolated membranes, but did increase both the rate and extent of reduction by NADPH in Triton-treated cells. 3. A cytochrome b was present also in the specific granule fraction of human neutrophils. Its Em (pH 7.0) was found to be -248 mV, very similar to that of the plasma-membrane cytochrome b. 4. The rate of oxidation of reduce cytochrome b-245 by air-saturated buffer, was determined by using stopped-flow techniques. In intact membranes t 1/2 for oxidation was 4.7 ms. This rate is sufficiently rapid to support the view that cytochrome b-245 is the oxidase in the respiratory burst of neutrophils. 5. Plasma-membrane cytochrome b of human neutrophils formed a complex with CO. At room temperature and 1 atm of CO approx. 40% of the cytochrome formed a complex; approx. 60% binding was measured at the increased concentration of dissolved CO achieved at 5 degrees C. The concentration of CO giving 50% binding was 1.18 mM.     

The development of cytochrome b-245 in maturing human macrophages.

Cytochrome b-245, the putative terminal component of the specialized cidal oxidase system of phagocytes, was measured in human monocytes in culture. There was a dramatic synthesis of the cytochrome, which increased by 27.3 +/- 2.0 pmol/day per 10(7) cells. This represents an increase of about 40%/day in the early stages and an overall 7-fold increase after 16 days. The protein content increased 3-fold over the same period, resulting in a doubling of the specific content of the cytochrome b. The newly synthesized cytochrome b was identified as that specifically located in the microbicidal oxidase electron-transport chain, as titration demonstrated that, at day 16 of maturation, 70% of the total membrane cytochrome b had a very low midpoint potential (-260 to -220 mV), characteristic of that found in this oxidase system. This cytochrome distributed with the plasma membrane on analytical subcellular fractionation, and a close relationship was observed between the maturation-induced increase in the concentration of this molecule and the capacity of the cells to produce superoxide.     

Phosphorylation of the subunits of cytochrome b-245 upon triggering of the respiratory burst of human neutrophils and macrophages.

Cytochrome b-245, the only clearly identified component of the microbicidal oxidase system of phagocytes, is a heterodimer consisting of a 23 kDa (alpha) and a 76-92 kDa (beta) subunit. This study was conducted to examine whether, in common with a number of proteins, the subunits of the cytochrome were phosphorylated upon activation of the oxidase. Both subunits were phosphorylated after activation of neutrophils or macrophages with phorbol myristate acetate or a phagocytic stimulus, although the time course of this process did not parallel that of the oxidase. Phosphorylation of these proteins was normal in cells from two patients with autosomal recessive chronic granulomatous disease, in whom phosphorylation of a 47 kDa protein is defective.     

Mechanism of the superoxide-producing oxidase of neutrophils. O2 is necessary for the fast reduction of cytochrome b-245 by NADPH.

A soluble oxidase from phorbol-stimulated pig neutrophils contained FAD and cytochrome b-245. A typical preparation produced 13.03 mol of superoxide (O2-.) X S-1 X mol of cytochrome b-1 (348 nmol X min-1 X mg of protein-1). In the aerobic steady state, cytochrome b was 8.9% reduced. Steady-state cytochrome b reduction was absent from extracts of unstimulated cells; Km values for NADPH, for O2-. production and cytochrome b reduction were similar. The calculated aerobic rate of cytochrome b reduction was equal to the measured rate of O2-. production in a variety of preparations and in the presence of a range of inhibitors. Under anaerobic conditions the rate was slow: O2 is apparently required for rapid electron flow into the oxidase complex. Cytochrome b is shown to be kinetically competent to act as part of the O2-.-generating complex.     

Isolation from neutrophil membranes of a complex containing active NADPH oxidase and cytochrome b-245

The lipid composition and fluidity of basolateral membranes prepared from the mucosa of the proximal, middle and distal thirds of the rat small intestine were determined. Fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and a series of anthroyloxy fatty acid derivatives, is decreased in the distal third as compared to the proximal segments. This pattern is similar to that described previously for microvillus membranes. The decrease in fluidity of the distal as compared to the proximal membranes results from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues. In the middle and distal thirds of the gut, the degree of saturation of the fatty acid residues is higher in microvillus as compared to basolateral membranes, accounting in part for the characteristically lower fluidity of the luminal membranes. The specific activity of the basolateral membrane (Na+ + K+)-dependent adenosine triphosphatase is significantly lower in the distal as compared to the proximal and middle thirds of the intestinal mucosa. Studies of the binding of [3H]ouabain indicate that this pattern results from fewer enzyme sites in the distal membranes.     

CO-reacting haemoproteins of neutrophils: Evidence for cytochrome b-245 and myeloperoxidase as potential oxidases during the respiratory burst

Room temperature, CO-difference spectra of intact rat polymorphonuclear leucocytes (neutrophils) revealed the presence of a number of CO-binding haemoproteins. Absorption maxima at 413, 540 and 570 nm were attributed to the CO-complex of cytochromeb-245 whereas an absorption maximum at 595 nm was assigned to the contribution from a myeloperoxidase complex, since an identical absorption maximum was observed in CO-difference spectra of purified myeloperoxidase in the presence of H₂O₂. Photochemical action spectra for the relief of CO-inhibited O₂ uptake revealed contributions from both cytochromeb-245 and myeloperoxidase. The potential of these two O₂- and CO-binding haemoproteins to function as oxidases during the respiratory burst is discussed.     

CD11b is a calcium-dependent epitope in human neutrophils.

Human neutrophils expressing complement receptor 3 (CR3) were treated with various concentrations (0.04-10 mM) of Ca2+/Mg(2+)-chelating agent EDTA and the expression of CD11b, the CR3 alpha chain antigenic epitope, was examined using monoclonal antibodies and flow cytometry. EDTA caused a dose-dependent decrease in the reactivity of two anti-CD11b monoclonal antibodies, Leu15 and IOM1. The reduced expression of CD11b in EDTA-treated cells was partly restored by the addition of Ca2+ ions whereas the addition of Mg2+ ions had no effect on CD11b level. The expression of the CR3 beta chain epitope, CD18, was markedly decreased only by 10 mM EDTA. These results suggest that the CD11b epitope may be associated with the Ca(2+)-binding domains of CR3 alpha chain and its recognition by antibodies depends on the presence of bound Ca2+.     

Purification and Some Properties of Cytochrome b-560 from Nitrosomonas europaea

Cytochrome b-560 was purified to an electrophoretically homogeneousstate from Nitrosomonas europaea. It showed absorption peaksat 427, 530 and 560 nm in the reduced form. Its molecular weightwas estimated to be 44,000 by SDS-polyacrylamide gel electrophoresisand the same value was obtained on the basis of the contentsof haem and protein. The cytochrome was not autoxidizable anddid not react with CO. ¹Present address: Tokyo Research Center, TOSOH Corporation,Hayakawa, Ayase-shi, Kanagawa 252, Japan ²Present address: Faculty of Integrated Arts and Sciences, HiroshimaUniversity, Higashisenda-machi, Hiroshima 730, Japan (Received March 23, 1988; Accepted June 2, 1988)     

Cytochrome b-559 may function to protect photosystem II from photoinhibition

Although cytochrome b-559 is an integral component of the photosystem II complex (PSII), its function is unknown. Because cytochrome b-559 has been shown to be both photooxidized and photoreduced in PSII, one of several proposals is that it mediates cyclic electron transfer around PSII, possibly as a protective mechanism. We have used electron paramagnetic resonance spectroscopy to investigate the pathway of photooxidation of cytochrome b-559 in PSII and have shown that it proceeds via photooxidation of chlorophyll. We propose that this photooxidation of chlorophyll is the first step in the photoinhibition of PSII. The unique susceptibility of PSII to photoinhibition is probably due to the fact that it is the only reaction center in photosynthesis which generates an oxidant with a reduction potential high enough to oxidize chlorophyll. We propose that the function of cytochrome b-559 is to mediate cyclic electron transfer to rereduce photooxidized chlorophyll and protect PSII from photoinhibition. We also suggest that the chlorophyll(s) which are susceptible to photooxidation are analogous to the monomer chlorophylls found in the bacterial photosynthetic reaction center complex.     

Studies of cytochrome b-245 translocation in the PMA stimulation of the human neutrophil NADPH-oxidase

The human neutrophil respiratory burst, activated by phorbol 12-myristate 13-acetate (PMA), results from specific receptor-ligand binding and activation of the NADPH-oxidase in the plasma membrane. The role of granule membrane constituents has been elucidated with neutrophils disrupted by nitrogen cavitation and then fractionated in Percoll gradients to resolve four postnuclear fractions: cytoplasm, light membranes or gamma fraction (site of the NADPH-oxidase), a light granule (beta) fraction containing putative constituents of the NADPH-oxidase (cytochrome b-245 and an associated flavoprotein), and a fraction of heavy granules. Cytochrome b-245 is localized to two pools of specific granules within the beta fraction as assessed by differing sedimentation in narrow Percoll gradients and translocates upon PMA-stimulation from one of these specific granule sub-pools to the plasma membrane where it exhibits no change in its midpoint redox potential. Translocation of cytochrome b-245 parallels O2-production initiated by PMA stimulation as assessed in the time course of each activity. The finding of increased amounts of the b cytochrome in cytoplast membranes relative to plasma membranes of unstimulated cells suggests that the cytoplasts, devoid of granules yet capable of O2-generation upon PMA-stimulation, are useful in assessing post-translocation events in the activation pathway of the NADPH-oxidase. These data support the hypothesis that translocation of NADPH-oxidase components from an intracellular granular pool contributes to respiratory burst expression.     

Cytochrome b-561 in sympathetic nerve terminal vesicles from rat vas deferens

The spectral properties of sympathetic nerve vesicles isolated from the vas deferens of the rat are similar to those of the bovine chromaffin granule membranes and bovine nerve trunk vesicles, indicating the presence of the specific cytochrome b-561. The cytochrome occurs only in the fractions containing nerve vesicles, thus suggesting usefulness as a marker enzyme.     

Role of cytochrome b-559 in arachidonic acid activation of resting human neutrophils

Whether or not cytochrome b-559 is a necessary component of NADPH oxidase activity in neutrophils is still controversial. In highly purified plasma membranes isolated from resting neutrophils and lacking cytochrome b, addition of arachidonic acid induced an NADPH oxidase activity. This activity was similar to that of plasma membranes isolated from phorbol myristate acetate (PMA)-stimulated cells which possessed cytochrome b. Addition of arachidonic acid to the latter plasma membranes did not alter the oxidase activity. It can be concluded that plasma membranes isolated from resting neutrophils have, in the presence of arachidonic acid, an NADPH oxidase activity similar to that of PMA-stimulated cells, except that it is independent of cytochrome b-559.     

The association of FAD with the cytochrome b−245 of human neutrophils

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b₋₂₄₅ at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b₋₂₄₅ the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b₋₂₄₅ and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b₋₂₄₅ of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b₋₂₄₅ and support a scheme for electron transport thus: [Formula: see text]     

The human fibroblast receptor for gp86 of human cytomegalovirus is a phosphorylated glycoprotein.

A human embryonic lung (HEL) cell receptor for gp86 of human cytomegalovirus that functions in virus-cell fusion was further characterized. Anti-idiotype antibodies that mimic gp86 were used to immunoprecipitate the 92.5-kDa fibroblast membrane receptor for gp86, which was preincubated with various endoglycosidases. The receptor, which has a pI ranging from 5.3 to 5.6, appears to be a glycoprotein with primarily N-linked sugar residues, some of which have high concentrations of mannose and some of which are complex oligosaccharides. Western blots (immunoblots) of electrophoretically transferred receptor incubated with various biotinylated lectins confirmed the presence of sugar moieties, including N-acetylglucosamine, glucose or mannose, and galactose, but not fucose or N-acetylgalactosamine. This gp86 receptor from uninfected HEL cells also incorporated radiolabeled phosphate from orthophosphoric acid, indicating that it is a constitutively phosphorylated receptor.     

A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain.

NADPH is a system in phagocytic cells that generates O2- and hydrogen peroxide in the endocytic vacuole, both of which are important for killing of the engulfed microbe. Dysfunction of this oxidase results in the syndrome of chronic granulomatous disease, characterized by a profound predisposition to bacterial and fungal infections. A flavocytochrome b is the site of most of the mutations causing this syndrome. The FAD and NADPH binding sites have been located on the beta subunit of this molecule, the C-terminal half of which showed weak sequence similarity to other reductases, including the ferredoxin-NADP reductase (FNR) of known structure. This enabled us to build a model of the nucleotide binding domains of the flavocytochrome using this structure as a template. The model was built initially using a novel automatic modeling method based on distance-matrix projection and then refined using energy minimization with appropriate side-chain torsional constraints. The resulting model rationalized much of the observed sequence conservation and identified a large insertion as a potential regulatory domain. It confirms the inclusion of the neutrophil flavocytochrome b-245 (Cb-245) as a member of the FNR family of reductases and strongly supports its function as the proximal electron transporting component of the NADPH oxidase.