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O T Jones

Publications and source records attributed to O T Jones.

At least 91 records · Page 5Linked to original sources
PubMed1984

Oxygen radical production by horse and pig neutrophils induced by a range of crystals.

The exposure of animal neutrophils to crystals implicated in joint inflammation induces superoxide and peroxide generation in a concentration and temperature dependent fashion. Active crystals were urate, hydroxyapatite, pyrophosphate and brushite; diamond and cholesterol were inactive. Cytochalasin B increased superoxide yield after addition of brushite, and inhibitors of the PMA induced respiratory burst blocked the crystal induced response. Addition of urate to anaerobic neutrophils causes the reduction of a b-type cytochrome, a likely component of the neutrophil superoxide generating system.

Animals↗
PubMed1983

The subcellular localization of ubiquinone in human neutrophils.

Ubiquinone-10 has recently been proposed as a component of the microbicidal oxidase system of neutrophil leukocytes [Crawford & Schneider (1982) J. Biol. Chem. 257, 6662-6668]. We have determined the subcellular localization of ubiquinone and could detect it only in the mitochondrial fractions. It was not enriched in phagolysosomes which were enriched in O2-. generating activity and cytochrome b-245. It is proposed that ubiquinone is unlikely to play a direct role in the electron transport chain located in the plasma membrane which functions to produce microbicidal oxygen radicals.

Centrifugation, Density Gradient↗
PubMed1983

The cytochrome b and flavin content and properties of the O2- -forming NADPH oxidase solubilized from activated neutrophils.

NADPH-dependent O2- -generating activity was extracted and partially purified from guinea pig polymorphonuclear leukocytes. The most active preparation generated 202.8 nmol O2- min/min per mg protein. This activity was 30-fold higher than that of extracts from resting cells, indicating that the activated state of the oxidase was retained after solubilization. The solubilization and purification of the enzyme activity were followed by a parallel solubilization and purification of cytochrome b. Spectroscopic studies showed that solubilized cytochrome b has an Em of -245 mV and binds CO to about 30%. Cytochrome b was reduced by NADPH in anaerobiosis at a low rate and was rapidly reoxidized by air. A correlation was found between the inhibition of O2- formation caused by the SH reagent p-chloromercuribenzoate and the alterations induced by this compound on the Em of cytochrome b. These observations strongly support the participation of cytochrome b in the catalytic activity of the solubilized NADPH oxidase. The enzyme preparations contained FAD, which was found to be associated both with NADPH oxidase and with diaphorase activities. The fraction with the highest O2- forming activity contained FAD and cytochrome b in a ratio of about 0.5:1. The participation of FAD in the electron transport from NADPH to O2 is supported also by the inhibitory effect exerted by quinacrine on O2- formation.

Animals↗
PubMed1983

Binding of dansyl propranolol to the (Ca2+ + Mg2+)-ATPase.

We have studied the binding of dansyl propranolol to lipid bilayers and to the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum. The fluorescence emission spectra for dansyl propranolol bound to the ATPase system can be fitted to the sum of three peaks, characteristic of probe bound to lipid and to protein and free in solution, respectively. Titrations show that binding to the lipid component of the ATPase system is comparable to binding to simple lipid bilayers. Binding constants obtained using fluorescence spectroscopy for binding to lipid bilayers agree with constants obtained from microelectrophoresis measurements. Binding to sites on the ATPase can be described either in terms of the aqueous concentration of dansyl propranolol or in terms of the mole fraction of dansyl propranolol in the lipid phase of the membrane. Both descriptions suggest extensive binding to annular sites at the lipid/protein interface of the ATPase. Binding at other sites on the ATPase might also be present. Binding of dansyl propranolol to the ATPase results in a marked inhibition of activity. At high Ca2+ concentrations, inhibition fits to a non-competitive model of inhibition, described by a Ki of 5 microM. We attribute this effect to binding at annular sites. At lower Ca2+ concentration, a decrease is observed in the apparent affinity of the ATPase for Ca2+ which can be attributed to a build-up of positive charge on the membrane as a result of binding.

Animals↗
PubMed1983

Changes in the cytochrome composition of Rhodopseudomonas sphaeroides grown aerobically, photosynthetically and on dimethyl sulphoxide.

Several strains and mutants of Rhodopseudomonas sphaeroides can be grown anaerobically in the dark in the presence of dimethyl sulphoxide as an electron acceptor. During adaptation to this fermentative mode of growth, two major c-type cytochromes are synthesized, one with Mr 45 000 and the second with Mr 20 000 and a midpoint potential of +120 mV. These cytochromes are barely detectable in membranes prepared from cells grown in aerobic or photosynthetic conditions. An electrophoretic method is presented for the detection of the b-type and c-type cytochromes of pigmented or unpigmented membranes. The method resolves three b-type cytochromes and four c-type cytochromes in membranes from aerobically and photosynthetically grown cells.

Aerobiosis↗
PubMed1983

Absence of cytochrome b-245 in chronic granulomatous disease. A multicenter European evaluation of its incidence and relevance.

The heme-containing protein cytochrome b-245 has been proposed as a primary component of the microbicidal oxidase system of phagocytes that normally generates superoxide-free radicals but when defective is associated with chronic granulomatous disease. We measured this cytochrome in granulocytes from 27 patients with chronic granulomatous disease and from 64 members of their families. It was undetectable in all 19 of the men in whom the defect appeared to be located on the X chromosome. Female relatives who were heterozygous carriers had reduced concentrations of the cytochrome and variable proportions of cells that were unable to generate superoxide; these two characteristics were closely related (r = 0.93 in the 16 mothers and 0.85 in all 24 carriers, P less than 0.001). In contrast, in all eight patients (seven women) with a probable autosomal recessive inheritance the cytochrome was present but nonfunctional. The properties tested, including midpoint potential, carbon monoxide binding, and organelle distribution, were normal, but the cytochrome did not undergo reduction on cellular stimulation. Thus, absence or malfunction of the cytochrome b-245 may be the causal molecular defect in chronic granulomatous disease, implicating it in the microbicidal oxidase system.

Adolescent↗
PubMed1983

A variant form of X-linked chronic granulomatous disease with normal nitroblue tetrazolium slide test and cytochrome b.

Chronic granulomatous disease was diagnosed in a boy who suffered from severe generalized infections. Family investigations revealed the inheritance of the disease to be X-linked. However, unlike other cases of X-linked chronic granulomatous disease, the membrane oxidase of the neutrophils from this patient was not totally defective and sufficient activity was left to result in a normal phorbol myristate acetate-stimulated nitroblue tetrazolium slide test. Also, unlike the usual findings in X-linked chronic granulomatous disease, cytochrome b was present in normal amounts in the neutrophils from this patient. The cytochrome was normal, judged from its midpoint potential of -245 mV and its ability to bind CO. It is thus apparent that X-linked chronic granulomatous disease may result from at least two different defects and that the phorbol myristate acetate stimulated nitroblue tetrazolium slide test fails to detect some cases.

Adolescent↗
PubMed1983

Effects of respiratory-burst stimuli on the flow properties of pig blood neutrophils.

Stimulants of the neutrophil respiratory burst, both soluble and crystalline, greatly inhibit the flow of cells through a Nuclepore filter with 5 micron diameter pores. The stimuli appear to reduce cell deformability by a mechanism independent of the activation of respiration, since inhibitors of the respiratory burst (N-phenyl maleimide, quercetin) are without effect on the change. It is suggested that stimuli reduce membrane deformability rather than cause aggregation, since slowing of flow is independent of cell density and is much less when the filter pore size is increased to 8 micron. The technique, recently used to study alterations in erythrocyte deformability, may be appropriate for investigating leucocyte flow in some clinical disorders.

Animals↗
PubMed1982

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase.

Quenching of the fluorescence of the (Ca2+ + Mg2+)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3 beta-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.

Animals↗
PubMed1982

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]

Cell Membrane↗
PubMed1982

Interaction of fatty acids with the calcium-magnesium ion dependent adenosinetriphosphatase from sarcoplasmic reticulum.

The fluorescence emission spectrum of dansylundecanoic acid is sensitive to the environment and appears at a lower wavelength when the fatty acid is bound to protein than when it is bound to phospholipid. When bound to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum, the emission spectrum can be resolved into separate components assigned to fatty acid bound to protein and to lipid. Efficiency of energy transfer from the tryptophan residues of the ATPase to dansylundecanoic is higher for protein-bound probe than for lipid-bound probe. Fluorescence titrations are consistent with three fatty acid binding sites per ATPase with a Kd of 7 microM, and these sites are postulated to occur at the protein-protein interface in ATPase oligomers. Fatty acid incorporated into the lipid component of the membrane appears to be bound outside the lipid annulus around the protein.

Animals↗
PubMed1982

The use of N-methylprotoporphyrin dimethyl ester to inhibit ferrochelatase in Rhodopseudomonas sphaeroides and its effect in promoting biosynthesis of magnesium tetrapyrroles.

N-Methylprotoporphyrin dimethyl ester inhibits ferrochelatase in isolated membranes of Rhodopseudomonas sphaeroides at low concentrations (around 10 nm). Full inhibition developed after a short lag phase. The inhibition was non-competitive with porphyrin substrate. Addition of inhibitor to growing cultures of Rps. sphaeroides caused a decrease (near 40%) in cytochrome content and a severe inhibition of ferrochelatase; the excretion of haem into the medium by cell suspensions was also severely inhibited. The addition of N-methylprotoporphyrin dimethyl ester to suspensions of photosynthetically competent Rps. sphaeroides Ga caused excretion of Mg-protoporphyrin monomethyl ester. When added to mutants V3 and O1, magnesium divinylphaeoporphyrin a5 monomethyl ester and 2-devinyl-2-hydroxyethylphaeophorbide a were excreted, with maximum effect at around 3 microM-inhibitor in the medium. The results are interpreted to suggest that the inhibitor decreases concentration of intracellular haem, which normally controls the activity of 5-aminolaevulinate synthetase. Unregulated activity of this enzyme leads to overproduction of protoporphyrin, which is diverted to the bacteriochlorophyll pathway. Further control operates at magnesium protoporphyrin ester conversion in normal cells.

Cell Membrane↗
PubMed1982

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.

Animals↗
PubMed1982

Development of cytochrome b and an active oxidase system in association with maturation of a human promyelocytic (HL-60) cell line.

The human HL-60 myeloid leukaemia cell line developed, during maturational changes induced by dimethyl sulphoxide, an enhanced capacity for phorbol myristate acetate- stimulated oxidative activity and acquired a cytochrome b. Titration of the absorbance at 559 nm at potentials of-190 to -370 mV indicated that this cytochrome had a very low potential, differentiating it from mitochondrial and endoplasmic reticulum cytochromes and identifying it as the cytochrome b(-245) that has been recently found in other phagocytic cells. Subcellular fractionation studies of mature HL-60 cells showed that cytochrome b had a dual distribution within the cell. The lighter peak of activity was associated with the plasma membrane markers, adenylate cyclase and receptors for the N- formal-L-methionyl-L-leucyl-L-phenylalanine (f-Met-Leu-Phe) peptide. The denser components localized with the mitochondria but were distinct from mitochondrial cytochromes because whereas the activity of cytochrome c oxidase fell during HL-60 cell maturation, that of this cytochrome b was markedly increased. Concentrations of myeloperoxidase were unrelated to activity of the oxidase system and decreased as the cell matured. The increase in the concentrations of cytochrome b with cellular maturation parallelled the increase in the stimulated nonmitochondrial respiratory activity of these cells. The turnover of the hexose monophosphate shunt of immature cells was increased by the oxidising agents, methylene blue and tert-butylhydroperoxide, indicating that these immature cells have stimulated nonmitochondrial respiratory activity by maturing HL-60 cells is associated with, and is probably dependent upon, the acquisition by these cells of the cytochrome b(-245) oxidase system.

Cell Line↗
PubMed1981

Light-dependent cytochrome P-450 changes in mung beans (Phaseolus aureus).

Maximum concentrations of microsomal cytochrome P-450 are present in 3-4 day-old mung beans (Phaseolus aureus). On illumination of dark-grown seedlings, cytochrome P-450 and later cytochrome P-450 undergo a rapid decrease in concentration in vivo, with an apparent half-time of about 6 h. Conversely light-grown seedlings, transferred to darkness, show a slow accumulation of cytochrome P-450, doubling time of about 30 h, with a later accumulation of cytochrome P-420. Microsomal cytochromes b559, b560.5 and b562.5 do not significantly alter on light-dark transitions. Possible functions for dark-induced cytochrome P-450 are discussed.

Cytochrome P-450 Enzyme System↗
PubMed1981

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.

Animals↗
PubMed1981

The cytochromes in microsomal fractions of germinating mung beans.

Detailed studies of microsomal cytochromes from mung-bean radicles showed the presence of cytochrome P-420, particularly in dark-grown seedlings, accompanied by smaller quantities of cytochrome P-450. Similar proportions of cytochrome P-420 to cytochrome P-450 were found spectrophotometrically in vivo with whole radicles and hypocotyls. Assayed in vitro, maximum concentrations of both cytochromes were attained after 4 days of growth, before undergoing rapid degradation. Illumination of seedlings stabilized cytochrome P-450 and decreased the amount of cytochrome P-420. Three b cytochromes were present in the microsomal fraction, namely cytochromes b-562.5 (Em + 105 +/- 23 mV), b-560.5 (Em + 49 +/- 13 mV) and b5 (Em - 45 +/- 14 mV), all at pH 7.0. Of the b cytochromes, cytochrome b5 alone undergoes a rapid degradation after day 4, Changes in cytochrome b concentrations were confined to the microsomal fraction: mitochondrial b cytochrome concentrations were unaltered with age. Protohaem degradation (of exogenous methaemalbumin) was detected in microsomal fractions of mung beans. The rates of degradation were highest in extracts of young tissue and declined after day 4. The degradation mechanism and products did not resemble those of mammalian haem oxygenase.

Cytochromes↗