Search PubMed⌕ Search

Biomedical subjects

O T Jones

Publications and source records attributed to O T Jones.

At least 73 records · Page 4Linked to original sources
PubMed1988

Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor.

Interactions between lipids and the nicotinic acetylcholine receptor from Torpedo californica have been measured in reconstituted membranes containing purified receptor and defined lipids. The ability of brominated lipids to partially quench the intrinsic fluorescence of the acetylcholine receptor has been exploited to monitor contacts between the protein and the surrounding lipid. Relative binding constants for lipid binding to the protein have been quantitatively determined by measuring quenching observed in mixtures of brominated and nonbrominated lipids by use of equilibrium exchange equations developed by London and Feigenson [London, E., & Feigenson, G. W. (1981) Biochemistry 20, 1939-1948] and by Simmonds et al. [Simmonds, A. C., Rooney, E. K., & Lee, A. G. (1984) Biochemistry 23, 1432-1441]. Dioleoylphosphatidylcholine and its dibromo derivative are the two principal lipids used in the reconstituted membranes to establish the quenching parameters. Competition studies between cholesterol and phosphatidylcholine indicate that cholesterol does not compete effectively for the phospholipid sites presumed to surround the membrane-embedded portions of the receptor (annular lipids). However, dibromocholesterol partially quenches the receptor and leads to additional quenching of receptor in pure dibromophosphatidylcholine membranes. The results are consistent with the presence of additional binding sites for cholesterol that are not accessible to phospholipids (nonannular sites). Similar results are obtained by using cholesterol hemisuccinate and its dibromo analogue, both of which can be introduced into membranes more easily than cholesterol because of their greater solubility in water. Fatty acids appear to compete for both annular and nonannular sites, and analysis of the quenching data suggests that there are 5-10 nonannular sites associated with the receptor. Cholesterol has been shown to play a critical role in both acetylcholine receptor structural stabilization and ion channel activity, and the results presented here provide additional information about cholesterol-receptor interactions.

Animals↗
PubMed1988

The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface Ig.

EBV-transformed B lymphocyte cell lines (EBV-BLCL) produce superoxide after stimulation with phorbol ester, a capacity unique among nonmyeloid cells. The superoxide producing system of EBV-BLCL (B cell oxidase) was compared with the phagocytic NADPH-oxidase and the relationship of the capacity to produce superoxide to the presence of the EBV-genome was analyzed. The two EBV-transformed B cell lines F1 and HELL generated superoxide in response to PMA (2.3 nmol/10(6) F1 cells x 1 h and 6.27 nmol/10(6) HELL cells x 1 h with 1 microgram/ml of PMA), whereas no superoxide release was detected with the EBV-positive Burkitt lymphoma line WIL-2 and the EBV-negative plasmocytoma line U-266. Also, F1 and HELL showed lucigenin-dependent chemiluminescence (CL) after PMA-treatment, whereas no CL responses were detected from WIL-2 or U-266. Further, F1 and HELL cells contained a low potential cytochrome b-245 (10.9 and 61.0 pmol/mg protein, respectively) and also a 45 kDa diphenylene-iodonium (DPI)-binding peptide, both components of the phagocytic NADPH-oxidase. In contrast, neither the cytochrome b-245 nor the 45 kDa DPI-binding peptide were detected in WIL-2 and U-266. In addition, DPI inhibited O2- production by PMA-stimulated EBV-BLCL and polymorphonuclear granulocytes. Further, F1 line cells showed superoxide dismutase-inhibitable lucigenin-dependent CL when triggered by protein A-bearing staphylococci (Cowan strain I) or by a mAb directed against human IgG in the presence of solid-phase goat anti-mouse-Ig antibody. From a panel of eight EBV-BLCL, only five responded with CL when exposed to protein A-bearing staphylococci, whereas all showed CL when treated with phorbol ester. Inasmuch as all eight EBV-BLCL possessed surface Ig and a "functional" oxidase, their differential response to cross-linking of surface Ig may be determined by differences in signal transduction. Superoxide production by EBV-BLCL appears thus related to expression of an electron transport chain structurally homologous, if not identical, with the "phagocytic" NADPH-oxidase. Apparently, the presence of EBV-genome in B cell lines does not per se lead to expression of this oxidase. This suggests that nontransformed B cells may, at a certain differentiation stage, also express a superoxide-generating chain. From the finding of stimulation of superoxide production of EBV-BLCL via surface Ig it appears possible that also Ag may be able to trigger such B cells to production of superoxide which might have an important role in the physiology of B cells.

B-Lymphocytes↗
PubMed1988

Haem synthesis during cytochrome P-450 induction in higher plants. 5-Aminolaevulinic acid synthesis through a five-carbon pathway in Helianthus tuberosus tuber tissues aged in the dark.

Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effective blockers of chlorophyll synthesis in artichoke tuber and Euglena cells exposed to light.

Allylglycine↗
PubMed1987

Induction of synthesis of components of the hydrogen peroxide-generating oxidase during activation of the human monocytic cell line U937 by interferon-gamma.

Increased amounts of cytochrome b-245 and a 45 kDa polypeptide component of the hydrogen peroxide-generating oxidase were detected in the human monocytic cell line U937 after incubation in interferon-gamma. The time course of increase in cytochrome b-245 paralleled the induction of oxidase activity. Induction of the 45 kDa component had a different time course, having a greater lag before increased amounts were seen.

Cell Line↗
PubMed1987

The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel.

The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.

Biological Transport↗
PubMed1987

The use of continuous assays to characterize the oxidative cyclase that synthesizes the chlorophyll isocyclic ring.

A continuous spectroscopic assay has been developed for magnesium protoporphyrin monomethyl ester oxidative cyclase, which records either the dark formation of both free and protein-bound magnesium phaeoporphyrin or, following flash illumination, its corresponding chlorin. The properties of the enzyme were studied in wheat etioplasts. When plastids were pre-illuminated in the presence of NADPH all endogenous protochlorophyllide was converted into chlorophyllide and the product of dark incubation with magnesium protoporphyrin monomethyl ester was protein-bound magnesium 2-vinyl phaeoporphyrin a5 monomethyl ester with either a vinyl or an ethyl group at position 4 of the macrocycle alone. Rates of chlorin production from magnesium protoporphyrin monomethyl ester (up to 1240 pmol/h per mg of protein) were adequate to support known rates of plant chlorophyll synthesis. The enzyme required NADPH and O2 and had an approximate Km of 0.5 microM for magnesium protoporphyrin IX monomethyl ester. Lipid-soluble metal-complexing agents inhibited enzyme activity: hydrophilic agents were ineffective. The strong inhibition of mycobactin suggested the involvement of iron ions. Zinc protoporphyrin monomethyl ester, but not copper or nickel or metal-free protoporphyrin monomethyl esters, was a substrate; magnesium protoporphyrin dimethyl ester was inhibitory. The activity of the enzyme was unchanged by prior greening of the plants. The activity in isolated etioplasts was very dependent upon intactness of the plastid structure.

Chlorophyll↗
PubMed1987

The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages.

Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible.

Animals↗
PubMed1986

Effects of lipids and long-chain alkyl derivatives on the activity of (Ca2+-Mg2+)-ATPase.

The ATPase activity of the (Ca2+-Mg2+)-ATPase reconstituted into bilayers of phosphatidylcholines depends on the fatty acyl chain length of the phospholipids. It is shown that the fluorescence response to Ca2+ of the ATPase modified with fluorescein isothiocyanate is also dependent on phospholipid structure and is interpreted in terms of a change in the equilibrium between two forms of the ATPase, E1 and E2. A kinetic scheme for the ATPase is presented in which ATPase activity is markedly dependent on the rate of the transition between two phosphorylated forms of the ATPase, E1'PCa2 and E2'PCa2, and it is postulated that changing the phospholipid structure changes this rate. The rate of dephosphorylation of the ATPase and the ATP dependence of the E1'PCa2-E2'PCa2 transition are also lipid dependent. Binding of oleyl alcohol causes large, lipid-dependent changes in ATPase activity, and these are interpreted in terms of changes in the rates of these same steps. Oleylamine, which has been shown to bind more strongly at annular sites than at nonannular sites, inhibits ATPase activity irrespective of lipid structure, whereas fatty acids, which bind less strongly at annular sites, only inhibit at high concentrations. Methyl oleate, which binds more strongly at nonannular sites than at annular sites, causes marked stimulation for the ATPase reconstituted with short-chain lipids.

Animals↗
PubMed1986

The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase.

NADPH-dependent superoxide production by the solubilized oxidase of neutrophils was inhibited 36% by diphenylene iodonium at a 1:1 stoichiometry with the enzyme flavoprotein content. Addition of diphenylene iodonium strongly inhibited the NADPH-dependent reduction of both FAD and cytochrome b-245 in steady-state kinetic experiments. Incubation of solubilized enzyme with diphenylene [125I]iodonium resulted in the specific labelling of a polypeptide of Mr 45,000. In the presence of NADPH the amount of label incorporated into the polypeptide was reduced. There was no difference in labelling between enzyme prepared from stimulated or unstimulated cells.

Animals↗
PubMed1985

Interactions of pyrene derivatives with lipid bilayers and with (Ca2+-Mg2+)-ATPase.

The intensities of fluorescence emission for pyrene and a number of its derivatives increase on binding to lipid bilayers and to the (Ca2+-Mg2+)-ATPase purified from rabbit muscle sarcoplasmic reticulum. The effect is particularly marked for the less water-soluble derivatives. Changes in intensity for monomer and excimer emission as a function of lipid concentration can be fitted to a simple model to obtain binding parameters. The number of binding sites per lipid is 0.2-0.4. For the ATPase system, at least two classes of sites are necessary to fit the data, one corresponding to the lipid component and one to sites on the ATPase. Excimer emission from the postulated sites on the ATPase is less marked than that from lipid. Pyrene-dodecanoic acid and pyreneundecyltrimethylammonium bromide, which bind to a large number of sites on the ATPase, cause marked inhibition of ATPase activity at high concentration. Pyrene and a number of water-soluble derivatives cause stimulation of the ATPase reconstituted with dimyristoleoylphosphatidylcholine and little inhibition and bind to a small number of sites on the ATPase. It is concluded that excimer emission from pyrene derivatives in systems containing proteins cannot be used to obtain reliable information about rates of diffusion in the lipid component of the membrane.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Interactions of hexachlorocyclohexanes with the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum.

Hexachlorocyclohexanes have been shown to inhibit the (Ca2+ + Mg2+)-ATPase of muscle sarcoplasmic reticulum reconstituted into bilayers of dioleoylphosphatidylcholine. However, for the ATPase reconstituted into bilayers of dimyristoleoylphosphatidylcholine, a pattern of activation at low concentration followed by inhibition at higher concentration is seen for hexachlorocyclohexanes and alkanes such as decane and hexadecane. The ATPase in sarcoplasmic reticulum vesicles is also inhibited by the hexachlorocyclohexanes. The effects of hexachlorocyclohexanes on activity are largely independent of concentrations of Ca2+ and ATP. Inhibition is more marked at lower temperatures. The hexachlorocyclohexanes quench the tryptophan fluorescence of the ATPase, and the quenching can be used to obtain partition coefficients into the membrane system. As for simple lipid bilayers, partition exhibits a negative temperature coefficient. Binding is related to effects on ATPase activity.

Animals↗
PubMed1985

Phagocytosis of brushite crystals by pig neutrophils.

Neutrophils from pig blood were disrupted by homogenisation or sonication and placed on an analytical sucrose gradient. Pig neutrophil azurophil granules were less dense than the specific granules, unlike those from neutrophils of most other mammalian species. Brushite crystals, which stimulate the respiratory burst in pig neutrophils, were found by electron microscopy to be phagocytosed. Membrane-limited vesicles containing crystals were obtained from a dense region of the sucrose gradient loaded with a homogenate of crystal treated cells. Intake of crystals involved preferential mobilisation of cytochrome b from the plasma membrane and the fusion of both specific and azurophil granules with the primary phagosome. Plasma membrane and granule markers appear in a crystal-containing region of the sucrose gradient when cells are treated with crystals. They are present in much lower concentration at this location in gradients from cells untreated with a crystal stimulus.

Animals↗
PubMed1984

Composition of partially purified NADPH oxidase from pig neutrophils.

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.

Animals↗
PubMed1984

The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.

Animals↗
PubMed1984

Membrane fluidity is not an important physiological regulator of the (Ca2+-Mg2+)-dependent ATPase of sarcoplasmic reticulum.

The (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum has been reconstituted into phospholipid bilayers of different fatty acyl chain length and degree of unsaturation, and ATPase activity was found to vary about 10-fold. Order parameters for bilayers of these lipids measured using the ESR probe 5-doxylstearic acid varied from 0.5 to 0.6 at 37 degrees C. There was no correlation between phospholipid order parameter and ATPase activity, and we conclude that phospholipid structure is a more important determinant of protein activity than is membrane fluidity.

Animals↗
PubMed1984

Ferrochelatase activity in the photosynthetic alga Cyanidium caldarium. Development of the enzyme during biosynthesis of photosynthetic pigments.

Dark-grown cells of the photosynthetic alga Cyanidium caldarium were shown to contain ferrochelatase activity, which increased markedly when the cells were induced to form pigments by exposure to light. Km values for the crude enzyme preparation were 14.8 microM and 6.5 microM for binding of Co2+ and deuteroporphyrin IX respectively.

Ferrochelatase↗