The subcellular distribution and some properties of the cytochrome <i>b</i> component of the microbicidal oxidase system of human neutrophils

Segal, Anthony W.; Jones, Owen

doi:10.1042/bj1820181

Cited by 134 publications

(43 citation statements)

References 37 publications

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“…A second peak at 420 nm also was observed, characteristic of cytochrome P-420. As described by others (37,38), both the membrane and granule fractions exhibited dithionite-reduced vs. oxidized difference spectra characteristic of cytochrome b (peaks at 429 nm and 559 nm). Granule fractions also exhibited a peak at 474 nm, characteristic of reduced myeloperoxidase (34) (Fig.…”

Section: Resultsmentioning

confidence: 95%

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Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Shak¹,

Goldstein²

1985

J. Clin. Invest.

128

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Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by w-oxidation. To characterize the enzyme(s) responsible for w-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 w-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 w-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a K, for LTB4 of 0.6 MM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent K. for NADPH = 0.5 AM; apparent K, for NADH = 223 MM). The LTB4 w-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by a-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB. w-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPHdependent cytochrome P450.

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Section: Resultsmentioning

confidence: 95%

“…There are at least four possible explanations to account for previous failures to detect cytochrome P450 in human PMN. First, the influence of carbon monoxide on difference spectra has been examined in only a few instances (37,(48)(49)(50). Second, PMN contain only small amounts of cytochrome P-450 (see below).…”

Section: Discussionmentioning

confidence: 99%

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Shak¹,

Goldstein²

1985

J. Clin. Invest.

128

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“…Monocytes aind neuitrophils were usecl at a concentrationi of 3 x 106 cells/ml. Latex particles were coated with humian IgG as described (16).…”

Section: Introductionmentioning

confidence: 99%

Role of oxygen in antibody-dependent cytotoxicity mediated by monocytes and neutrophils.

Borregaard¹,

Kragballe²

1980

J. Clin. Invest.

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A B S T R A C T The antibody-dependent cell-mediated cytoxicity (ADCC) by human monocytes and neutrophils was investigated by measuiriing the release of 'chromate from prelabeled erythrocytes coated with immunoglobulin G. ADCC was found to be positively correlated to phagocytosis of 51Cr-labeled erythrocytes and to the postphagocytic events of the effector cells, activation of the hexose monophosphate shunt, and degranulation.Exclusion of oxygen from the incubatioin media halved the ADCC by both cell types without affecting phagocytosis or degranutilation. Likewise, ADCC by cells from patients suffering from chronic granulomatous disease (CGD) was only half the intensity of ADCC by cells from normals. Inhibitors of mitochondrial respiration were without depressing effect on ADCC. Azide, which in addition to its blocking action on oxydative phosphorylation also inhibits catalase and myeloperoxidase, resulted in a -40% stimulation of ADCC by cells from normals but was without effect oIn ADCC by cells from CGD patients. The hydroxyl radical scavenger, mannitol, significantly depressed ADCC by cells from normals (P < 0.01) but was without effect on cells from CGD patients. Azide and mannitol also were without effect on ADCC by normal cells when oxygen was excluded.In a xanthine-xanthiine oxidase system, erythrocytes were effectively lysed. This lysis was inhibited by catalase, superoxide dismutase, and mannitol. When comparable concentrations of gltucose oxidase were tused no lysis was observed. H202 either alone or in combination with azidle did not lyse erythrocytes.It is suggested that ADCC by 1)oth monocytes and neutrophils is partly dependent on the generation of hydroxyl ra(licals by the effector cells.

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“…Human neutrophils were isolated from buffy coats (supplied by the South West Regional Transfusion Service, Southmead Hospital, Bristol) as previously described by Segal and Jones [25]. Membrane and cytosol fractions were prepared from resting neutrophils by differential centrifugation of disrupted cells as described for fibroblasts.…”

Section: Isolation Of Membranes and Cytosolmentioning

confidence: 99%

The functional expression of p47‐phox and p67‐phox may contribute to the generation of superoxide by an NADPH oxidase‐like system in human fibroblasts

et al. 1994

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Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to date on cultured human fibroblasts have primarily concerned the identification of cytochrome b558, whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47-phox and p67-phox. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47-phox, p67-phox and p22-phox. Expression of the gp91-phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47-phox antibody (JW-1), identified a 47 kDa protein.Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min//tg membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min//zg membrane protein). These data indicate that the expression of p47-phox and p67-phox by human fibroblasts may contribute to the cells' generation of superoxide.

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The subcellular distribution and some properties of the cytochrome b component of the microbicidal oxidase system of human neutrophils

Cited by 134 publications

References 37 publications

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Role of oxygen in antibody-dependent cytotoxicity mediated by monocytes and neutrophils.

The functional expression of p47‐phox and p67‐phox may contribute to the generation of superoxide by an NADPH oxidase‐like system in human fibroblasts

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