Cytochrome b-245 from human neutrophils is a glycoprotein.
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ABSTRACT:
SUBMITTER: Harper AM
PROVIDER: S-EPMC1144906 | biostudies-other | 1985 May
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC1153509 | biostudies-other
Project description: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]
| S-EPMC1154028 | biostudies-other
Project description: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.
| S-EPMC1163001 | biostudies-other
Project description: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.
| S-EPMC1153469 | biostudies-other
Project description: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.
| S-EPMC1158375 | biostudies-other
Project description: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.
| S-EPMC1149233 | biostudies-other
Project description: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.
| S-EPMC1147335 | biostudies-other
Project description: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.
| S-EPMC1144791 | biostudies-other
Project description:BackgroundThe association between short stature, undernutrition and the risk to cardiovascular disease has been clinically established. Genetic factor, particularly the variants in cytochrome b-245 alpha chain (CYBA) gene, which alter the formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase level, might affect arterial function. This study aimed to observe the association between single nucleotide variants (SNVs) of the CYBA gene and the arterial function of short stature children to understand the reason why some people with short stature develop cardiovascular disease.MethodsA total of 142 genomic deoxyribonucleic acid (DNA) samples have been collected from short stature children in Brebes, Central Java, Indonesia. Four common single-nucleotide polymorphisms (SNPs): C242T (rs4673), A640G (rs1049255), -930A>G (rs9932581) and *49A>G (rs7195830) in the CYBA gene were examined using TaqMan allelic discrimination assay. The arterial function was measured using transthoracic echocardiography and described as aortic stiffness and distensibility index. Statistical analysis was done to find a significant difference in arterial function between genotypes of each SNV.ResultsA P-value of < 0.05 was considered significant. In rs9932581 (-930A>G) of CYBA gene, the subjects with GG genotype were found to have significantly lower arterial stiffness and higher distensibility compared to AA and AG genotypes. No significant difference was found in the other SNVs.ConclusionThe GG genotype in rs9932581 of the CYBA gene might have a protective effect on cardiovascular disease in short stature children.
| S-EPMC8683098 | biostudies-literature
Project description:Cytochrome b-245 from neutrophil plasma membranes contains two types of subunit with apparent molecular masses from gel electrophoresis in the presence of SDS of 23 kDa and 76-92 kDa. Radiation-inactivation analysis revealed a single-exponential decay process for the visible absorption of the haem chromophore in the membrane, corresponding to a molecular mass of 21 +/- 5 kDa for the haem-containing polypeptide chain. Sedimentation equilibrium of the cytochrome solubilized by the detergent Triton N101 showed that the protein was polydisperse, with a molecular mass of approx. 350 kDa for the smallest detectable species. In another detergent, n-octyl beta-O-glucopyranoside (octyl glucoside), the molecular mass of the haem-containing particle was found to be 20-30 kDa. Thus the quaternary structure of the protein breaks down in this detergent. The haem group is inferred to be attached to the smaller subunit.
| S-EPMC1133673 | biostudies-other