Biomedical subjects

P Bellavite

Publications and source records attributed to P Bellavite.

At least 73 records · Page 4Linked to original sources

PubMed1987

Modification of the glyoxalase system during the functional activation of human neutrophils.

The glyoxalase system catalyses the metabolism of methylglyoxal to D-lactic acid, via the intermediate S-D-lactoylglutathione. It is present in human neutrophils and undergoes a significant modification during functional activation--induction of chemotaxis, phagocytosis and degranulation. During the activation of neutrophils with serum-opsonised zymosan and the tumour-promoting phorbol diester 12-O-tetradecanoylphorbol 13-acetate, the activity of glyoxalase I increases and the activity of glyoxalase II decreases by 20-40% of their activities in resting cells, in the initial 10 min of the activation period. Determination of the Michaelis constant, Km, and the apparent maximum velocity, Vmax, for these enzymatic reactions indicates that the change in activity is due to a non-competitive activation and inhibition of glyoxalase I and glyoxalase II, respectively. This is consistent with a modification of the glyoxalase enzyme protein during the activation response. This modification occurs under aerobic and anaerobic incubation conditions. The concentration of S-D-lactoylglutathione increases approx. 100% of the resting cell concentration during the initial 10 min of the activation period. The presence of S-D-lactoylglutathione in neutrophils may be related to its ability to stimulate microtubule assembly.

Cell Membrane↗

PubMed1987

S-D-lactoylglutathione in resting and activated human neutrophils.

Zymosan particles opsonised with human serum factors functionally activate human neutrophils and induce a substantial modification of the human neutrophil cytosolic glyoxalase system. The activity of glyoxalase I increases and the activity of glyoxalase II decreases by 20-40% of their resting cell activities during the initial 10 min of activation. The cellular concentration of the glyoxalase intermediate S-D-lactoylglutathione increases by ca. 100% of resting cell levels during this period. This modification may be related to the ability of S-D-lactoylglutathione to stimulate the assembly of microtubules.

Enzyme Activation↗

PubMed1987

Studies on the nature and activation of O2- -forming NADPH oxidase of leukocytes. II. Relationships between phosphorylation of a component of the enzyme and oxidase activity.

The activation of O2- -formation by neutrophil NADPH oxidase is associated with phosphorylation of several membrane and cytosolic proteins. In the membranes a phosphoprotein of 32 kDa belonging to the NADPH oxidase-cytochrome b-245 system (P. Bellavite et al., Free Rad. Res. Commun., 1, 11 (1985] showed the highest relative increase of 32Pi incorporation. Concomitant with the phosphorylation, a shift of the apparent molecular mass of the protein from 31 to 32 kDa occurred. The time-course, the sensitivity to trifluoperazine and the dose-dependence of phosphorylation were similar to those of O2- forming activity, except that the latter showed a longer lag-time than the former. The increase of the 32 kDa phosphoprotein was also comparable to the kinetics of cytochrome b-245 reduction by anaerobically activated neutrophils. The phosphorylation and the NADPH oxidase were triggered by various stimulants including phorbol myristate acetate, opsonized zymosan, arachidonic acid and sodium fluoride. With arachidonic acid the O2- formation was highly active but the phosphorylation was low. With fluoride the enzyme activity was reversible upon removal of the stimulant but the phosphorylation of the 32 kDa peptide was not reversible. Neutrophils treated with PMA at 17 degrees C showed phosphorylation but not activation. The results indicate that phosphorylation of a component of NADPH oxidase is a fundamental but probably not sufficient event in the activation mechanism of the enzyme.

Anaerobiosis↗

PubMed1986

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.

Cell Membrane↗

PubMed1986

Molecular basis of macrophage activation. Expression of the low potential cytochrome b and its reduction upon cell stimulation in activated macrophages.

The expression of the novel b-type cytochrome, which is part of the superoxide anion (O2-)-generating system in phagocytes, has been investigated in population of mouse peritoneal macrophages heterogeneous in their capability to produce O2-). Reduced minus oxidized difference spectra of intact cells showed the appearance of a b-type cytochrome with major peaks in the alpha region at 558 to 559 nm and in the gamma region at 426 to 428 nm. Resident peritoneal macrophages, as well as thioglycollate broth-elicited and Corynebacterium Parvum-activated macrophages and neutrophils expressed about 50 pmol cytochrome b/10(7) cells. In intact macrophages and neutrophils, Na-dithionite reduced greater than 75% of the cytochrome b measurable in disrupted cells. No correlation was found between capability to produce O2-) by different population of macrophages and their content of cytochrome b. When stimulated in strictly anaerobic conditions with phorbol myristic acetate, macrophages activated in vivo by i.p. injection of Corynebacterium Parvum reduced approximately 40% of their total cytochrome b. In resident peritoneal macrophages that produced five times lower amounts of O2-, cytochrome b reduction was instead undetectable. Potentiometric properties of cytochrome b was investigated in macrophage subcellular particles. Both resident and Corynebacterium Parvum-activated macrophages revealed the presence of b chromophores with very low potentials of -255 and -244 mV, respectively, whose content was not different in the two populations. These results show that resident and activated macrophages express the same amount of cytochrome b, but upon stimulation with PMA, activated macrophages recruit a higher number of cytochrome b molecules in parallel with an enhanced production of O2-.

Animals↗

PubMed1986

Respiratory response of phagocytes: terminal NADPH oxidase and the mechanisms of its activation.

The chemical composition, properties and activation mechanism of the O2(-)-forming NADPH oxidase of phagocytes were investigated, using partially purified enzyme preparations. Highly active NADPH oxidase was extracted as an aggregate of high Mr from the membranes of neutrophils and macrophages. The enzyme complex contained phospholipids and cytochrome b-245, very little FAD and almost no quinones or NAD(P)H-dye reductase activity. The purification of a polypeptide with a relative molecular mass of 31 500 strictly paralleled the purification of NADPH oxidase, suggesting that this polypeptide is a component of the enzyme. This protein was identified as cytochrome b -245 after dissociation of the proteolipid complex and purification of the cytochrome moiety. The 31 500 Mr protein was phosphorylated in enzyme preparations from activated but not from resting cells. The results indicate that: cytochrome b-245 is a major component of NADPH oxidase; the involvement of NAD(P)H dye reductases in the O2(-)-forming activity is questionable; the cytochrome b-245: FAD ratio in the enzyme complex is much higher than that indicated in crude preparations; the Mr of pig neutrophil cytochrome b-245 is 31 500; the activation of the O-2-forming system involves a process of phosphorylation of cytochrome b-245.

Animals↗

PubMed1986

Monoclonal antibodies to a particulate superoxide-forming system stimulate a respiratory burst in intact guinea pig neutrophils.

Monoclonal rat antibodies were produced against a subcellular preparation of phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig neutrophils that retains NADPH-oxidase activity. Two antibodies, 1A10.4 and IG4, were isolated that bind to a surface antigen restricted to guinea pig neutrophils from bone marrow and peritoneal exudate and to macrophages and that trigger a respiratory burst in neutrophils in the presence of cytochalasin B. Intact antibody 1A10.4, subclass IgG2c, can trigger superoxide anion release directly; F(ab')2 fragments of 1A10.4 and intact IG4 require further cross-linking by F(ab')2 fragments of anti-rat immunoglobulin antibody. Both antibodies recognize the same antigen, a proteolipid of apparent molecular mass 10 kDa. Immunoprecipitation of solubilized oxidase activity with 1A10.4 brings down this activity as part of a macromolecular complex. Surface expression of the antigen is increased on treatment of cells with both PMA and cytochalasin B. 1A10.4 also triggers release of the granule enzyme beta-glucuronidase. Triggering of a respiratory burst by the antibodies appears distinct from the PMA and fMet-Leu-Phe signalling systems. These studies indicate that the antigen defined by antibodies 1A10.4 and IG4 becomes associated with the superoxide anion-generating system of neutrophils but may play a more general role in signal transduction in phagocytic cells.

Animals↗

PubMed1985

Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (-247 mV) cytochrome b.

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be -247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low Em7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.

Animals↗

PubMed1985

Protein kinase C phosphorylates a component of NADPH oxidase of neutrophils.

A protein of 31.5 kDa belonging to the NADPH oxidase of neutrophils was phosphorylated following stimulation of the cells with phorbol myristate acetate. The same protein was phosphorylated in vitro in the presence of cytosol and of Ca2+ and phosphatidylserine. The phosphorylation in vitro of the 31.5 kDa protein was increased by phorbol myristate acetate and was inhibited by trifluoperazine. The data are compatible with an involvement of protein kinase C in the activation of NADPH oxidase.

Animals↗

PubMed1985

Intensity and kinetics of the respiratory burst of human neutrophils in relation to receptor occupancy and rate of occupation by formylmethionylleucylphenylalanine.

Studies on the relationship between the binding of fMet-Leu-Phe and the respiratory response in human neutrophils have been carried out under two different conditions of stimulus presentation, i.e., instantaneously and over a period of time. The main findings are as follows (1) Under the first condition the activation of the respiratory response reaches the maximum value very quickly, when the receptor occupancy is less than 20% that at equilibrium. After reaching this maximal value, the activated respiration progressively decreases, while the specific binding of the stimulant continues until equilibrium. (2) Under the second condition, i.e., when the stimulus to neutrophils is presented over a time of 1, 2 or 4 min, the respiratory response (and also the secretory one) is depressed or absent, and the initial rate of the binding (initial Vass) is lower, but the maximal values of the receptor occupancy at equilibrium and of the rate of receptor occupation (maximal Vass) are similar and only slightly lower than those reached under the condition of instantaneous presentation of the stimulus. (3) This form of desensitization is specific for fMet-Leu-Phe and does not consist of the inactivation of the target (NADPH oxidase), since neutrophils desensitized by the slow presentation of the peptide are able to respond to a second challenge with other stimulants. These results indicate that: (1) the efficacy of the stimulus-receptor complexes is short-lived; (2) the intensity of the respiratory response is dependent on the rate of reaching a threshold of binding; (3) when this initial rate is slow, owing to the slow presentation of the stimulus, a specific desensitization takes place, indicating the existence of a molecular mechanism, linked in some way to the initial rate of binding, that modulates the capacity of the stimulus-receptor complexes to transduce signals for cell responses. The physiological role of this type of desensitization is discussed.

Adult↗

PubMed1985

Mechanism of production of toxic oxygen radicals by granulocytes and macrophages and their function in the inflammatory process.

The paper deals with 1) the features of the respiratory burst (increase of the respiration with production of O2 metabolites, O2-, H2O2, OH) of the inflammatory cells; 2) the factors responsible for its activation; 3) the methods for its measurement; 4) the molecular events which take place at the level of the plasma membrane following the interaction between the stimuli and the cell surface (the Ca++ changes, the modification of membrane potential, the activation of phospholipid turnover) and the hypothesis of the activation of the protein kinase C; 5) the nature of the NADPH oxidase whose activation is responsible for the respiratory burst and the production of O2 metabolites; 6) the defensive, toxic, proinflammatory and modulatory effects due to the reactivity of the oxygen metabolites.

Animals↗

PubMed1985

Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme.

Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.

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

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

NADPH-dependent O2- forming activity was extracted with deoxycholate from subcellular particles of guinea-pig neutrophils following stimulation with phorbol myristate acetate. The solubilized enzyme was purified by chromatography on Ultrogel AcA22, by isopycnic glycerol density gradient centrifugation and by treatment with 0.4 M NaCl. This procedure yielded a high-molecular-weight complex containing phospholipids, cytochrome b-245 and NADPH oxidase activity. Cytochrome b was found to be purified to the same extent as NADPH oxidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the various purification fractions showed a progressive enrichment of a band whose molecular weight is 3.2 X 10(4). The enrichment of this protein band paralleled those of NADPH oxidase activity and of cytochrome b, indicating that it is a component of the oxidase system. The possibility that this band corresponds to either cytochrome b or a flavoprotein/cytochrome b complex is considered.

Animals↗

PubMed1984

NADPH oxidase of neutrophils forms superoxide anion but does not reduce cytochrome c and dichlorophenolindophenol.

Superoxide (O-2) production by partially purified NADPH oxidase from guinea pig neutrophils was markedly increased when the cells were activated by exposure to phorbol-myristate acetate. On the contrary, NADPH-dependent cytochrome c and 2,6-dichlorophenolindophenol (DCIP) reductase activities in preparations from resting and activated neutrophils were similar. The apparent Km values for NADH and NADPH of the reductase activities were different from those of the O-2 producing enzyme. The electron acceptors did not inhibit the oxygen consumption by NADPH oxidase in the presence of superoxide dismutase. Even in anaerobiosis the oxidase failed to reduce cytochrome c and DCIP. These results suggest that NAD(P)H-dependent dye reductase activities are not involved in the electron transport system responsible for the O-2 production by neutrophils.

2,6-Dichloroindophenol↗

PubMed1984

Chronic granulomatous disease in two sisters.

Two sisters with chronic granulomatous disease (CGD) have been studied. The diagnosis was suggested by the histopathological findings from the spleen and lymph nodes of the proband and confirmed by the low values obtained in the following tests performed on polymorphonuclear leukocytes (PMN): chemiluminescence, nitroblue tetrazolium (NBT) reduction, killing of Staphylococcus aureus, and O2- production. NADPH oxidase activity was not detected in the homogenates of the patients' PMN but cytochrome b was normally present. In addition, PMN depolarization induced by phorbol-myristate acetate was absent, thus suggesting a defect of the activation mechanism of the respiratory enzyme. The normal depolarization induced by ouabain indicated that the membrane polarity regulated by the Na/K pump in the patients' cells was not affected. The low, but not completely absent, respiratory activity of the patients' PMN could suggest an X-linked mode of inheritance with incomplete Lyonization. From a clinical point of view, one sister had mild symptoms whereas the other was almost symptomless, thus confirming once more the heterogeneity of CGD syndrome.

Adolescent↗

PubMed1983

Relationship between the binding of N-formylmethionylleucylphenylalanine and the respiratory response in human neutrophils.

The results presented in this paper demonstrate that the chemotactic peptide N-formylmethionylleucylphenylalanine (f-Met-Leu-Phe) is rapidly inactivated by the products of the respiration of human neutrophils stimulated by the peptide itself. The process of inactivation is impeded by the addition of inhibitors of myeloperoxidase (KCN, NaN3), of catalase, of methionine but not by the addition of superoxide dismutase, indicating that the mechanism of inactivation is the oxidation of methionine residue by myeloperoxidase-H2O2-halide system. The oxidation of the peptide causes the rapid cessation of the respiratory burst, since the sulfoxide derivative loses its ability to bind the specific receptors of neutrophil surface and, hence, its biological activity. The comparison between the time course of the binding of f-Met-Leu-[3H]Phe to the specific receptors and the rate of the respiratory response of neutrophils in the presence and in the absence of the process of peptide oxidation was used to investigate the mechanism of the activation of the respiratory burst by the peptide-receptor complexes. In conditions where the inactivation of the stimulatory agent takes place the stimulated respiration slows down and resumes the resting state shortly after the cessation of the binding, although a substantial amount of the peptide remains bound to the specific receptors. In conditions where the degradation of the peptide does not occur the binding of the peptide and the respiratory burst continue for a longer period of time, but the rate of the respiration, calculated in terms of the instantaneous velocity (Vist), is not correlated to the amount of the ligand bound to the membrane receptors measured at various times, indicating that a summation of the effects of the ligand-receptor complexes does not occur as they form. These findings demonstrate, as far as the respiratory response is concerned, that the biological activity of the peptide-receptor complexes is short-lived and that continuous de-novo receptor occupancy is necessary for the maintenance of the activated respiration.

Chemotactic Factors↗

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↗