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J T Curnutte

Publications and source records attributed to J T Curnutte.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase.

Human neutrophils and other phagocytes generate superoxide anion (O2-) as a means of destroying ingested microorganisms. O2- is produced by an NADPH-consuming oxidase composed of membrane and cytosolic components. Activation of the NADPH oxidase is absolutely dependent upon GTP, indicating the requirement for a GTP-binding protein in this process. We have utilized a five-step chromatographic procedure to isolate a GTP-binding protein from human neutrophil cytosol which can stimulate NADPH oxidase activity in a cell-free assay. Oxidase enhancing activity was shown to coisolate with this GTP-binding component, which was purified to apparent homogeneity. The GTP-binding protein was identified as Rac 2 by immunological reactivity and amino acid sequencing. Thus, Rac 2 appears to be a third cytosolic component required for human neutrophil NADPH oxidase activation. Recombinant Rac 2 was shown to bind guanine nucleotides in a Mg(2+)-dependent fashion. GDP dissociation rates were determined and shown to be regulated by the free Mg2+ concentration. Rac 2 was found to possess the highest rate of intrinsic GTP hydrolysis of any of the characterized members of the Ras superfamily. The biochemical properties of Rac 2 indicate it is likely to be subject to regulatory cofactors in vivo.

Chromatography, Affinity↗
PubMed1992

Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A.

Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane-derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b-negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

Cytochrome b Group↗
PubMed1992

Stabilization of human neutrophil NADPH oxidase activated in a cell-free system by cytosolic proteins and by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membrane and cytosol by anionic amphiphiles such as sodium dodecyl sulfate and arachidonate (McPhail, L. C., Shirley, P. S., Clayton, C. C., and Snyderman, R. (1985) J. Clin. Invest. 75, 1735-1739; Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743; Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221). Herein, the activity thus obtained is shown to be very labile at 37 degrees C. The rate of inactivation varied inversely with cytosol concentration. The stabilizing factor(s) was destroyed by heat and trypsin, indicating that it is protein in nature. Whereas cytosol from normal cells and from a chronic granulomatous disease patient lacking p67phox stabilized the oxidase activity, that from a chronic granulomatous disease patient lacking p47phox did not. Also, dialdehyde NADPH-treated cytosol showed no stabilizing effect, indicating that p47phox and a putative NADPH-binding component both participate in stabilization. The mechanism of inactivation was further explored by examining the stabilizing effect of agents that can act as chemical cross-linkers. Of several tested, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was the most effective, but others that utilize different chemical mechanisms were also partially effective. EDC extended the half-life at 37 degrees C from 2 to 120 min, protected against the inactivating effects of Triton X-100 and high salt, and did not affect the Km for NADPH. Stabilization required prior activation in the presence of both cytosol and membrane; and EDC treatment of cytosol, membrane, or a mixture of the two prior to the addition of sodium dodecyl sulfate failed to induce stabilization. EDC eliminated the requirement for the continuous presence of cytosol and activator. Dialysis did not cause a loss in activity, whereas control activity was diminished with dialysis and was largely restored with added sodium dodecyl sulfate. In the absence of EDC, the separation of cytosol from the membrane fraction resulted in a significant loss of activity, which was largely restored by the addition of cytosol. However, EDC treatment allowed the isolation of a nearly fully active oxidase in the membrane fraction, the activity of which was not influenced by added cytosol. These results support a model in which the active NADPH oxidase consists of a dissociable complex among membrane and cytosolic components and indicate that the longevity of the activated state requires continuous association of these components.

Cell Membrane↗
PubMed1992

Absolute requirement for GTP in activation of human neutrophil NADPH oxidase in a cell-free system: role of ATP in regenerating GTP.

Guanine and/or adenine nucleotides appear to be involved in the activation of the superoxide-generating NADPH oxidase of phagocytic cells. Their precise roles, however, are unclear, as much of the evidence for their involvement comes from experiments in which nucleotides have been added to complex systems already rich in both endogenous nucleotides and enzymes capable of interconverting them. To circumvent this problem we have examined the role of nucleotides in neutrophil NADPH oxidase activation by using a cell-free system in which adenine and guanine nucleotide concentrations were carefully controlled and monitored by (i) depletion of endogenous nucleotides by extensive dialysis and charcoal treatment; (ii) reconstitution of the depleted system with reagents analyzed for purity; and (iii) measurement of nucleotide levels in cytosol preparations and in oxidase reaction mixtures by HPLC analysis. In contrast to previous reports that have demonstrated only a several-fold enhancement of oxidase activity by GTP or its analogs, we have shown that oxidase activation was absolutely dependent upon GTP in reactions containing dialyzed cytosol in which the total endogenous nucleotide levels were reduced by greater than 99.5%. Kinetic studies revealed that GTP is required at or before the rate-limiting step in oxidase activation. Two nonhydrolyzable analogs of GTP, guanosine 5'-(gamma-thio)triphosphate and guanylyl imidodiphosphate, were even more active than GTP, suggesting the involvement of one or more GTP-binding proteins. In contrast, ATP was neither necessary nor sufficient for oxidase activation. If reaction mixtures were contaminated with GDP and/or GMP, however, ATP (but not its nonhydrolyzable analog adenylyl imidodiphosphate) could indirectly support oxidase activation by means of endogenous enzymes that catalyze the ATP-dependent conversion of GMP and GDP to GTP.

Adenine Nucleotides↗
PubMed1992

Prolonged recombinant interferon-gamma therapy in chronic granulomatous disease: evidence against enhanced neutrophil oxidase activity.

Recombinant interferon-gamma (rIFN-gamma) therapy has become an effective form of prophylaxis for patients with chronic granulomatous disease (CGD). Preliminary studies with CGD suggested that rIFN-gamma treatment enhanced phagocyte oxidase activity and increased superoxide (O2-) production. We evaluated several aspects of neutrophil NADPH oxidase activity in 19 CGD patients (representing all four known types of CGD) receiving prolonged rIFN-gamma therapy (6 to 27 months). In contrast to earlier studies, we failed to detect any improvement in neutrophil NADPH oxidase activity in 18 of the 19 CGD patients as determined by (1) intact cell O2- production (continuous assay), (2) nitroblue tetrazolium (NBT) staining, (3) cytochrome b558 spectroscopy, and (4) activity levels of cytosol and membrane oxidase components using a cell-free activation system. One patient with a variant form of X-linked CGD had a transient increase in neutrophil O2- production following 3 months of rIFN-gamma therapy. However, this was not sustained, and was not associated with any change in cytochrome b levels. In some patients, rIFN-gamma therapy was associated with the appearance of a small subset of circulating monocytes (1% to 20%) that were NBT-positive. Although the functional significance of this monocyte subpopulation needs to be determined, these results suggest that one possible mechanism by which rIFN-gamma may benefit CGD patients is by partially correcting the respiratory burst defect in a subset of monocytes. We conclude that the clinical benefit of prolonged rIFN-gamma therapy in the vast majority of CGD patients is not due to enhanced neutrophil NADPH oxidase activity. The mechanism of action of rIFN-gamma in most CGD patients remains unknown.

Child↗
PubMed1992

Identification of a thermolabile component of the human neutrophil NADPH oxidase. A model for chronic granulomatous disease caused by deficiency of the p67-phox cytosolic component.

Mild heating of human neutrophils inactivates the respiratory burst oxidase, producing a defect in superoxide production and bacterial killing comparable to that seen in patients afflicted with chronic granulomatous disease (CGD). We have now investigated the mechanism and specificity of this inactivation by examining the effect of mild heating on the known oxidase components: the membrane-bound subunits of the cytochrome b558 (gp91-phox and p22-phox) and the two cytosolic oxidase factors (p47-phox and p67-phox). Heating (46 degrees C for 7.5 min) caused intact neutrophils to lose greater than 85% of their capacity to produce superoxide, a defect which was localized to the cytosolic, but not the membrane, fraction. Complementation studies with CGD cytosols deficient in either p47-phox or p67-phox suggested that the defective component of heat-inactivated cytosol was p67-phox. This was confirmed by experiments showing that recombinant p67-phox, but not p47-phox, exhibited lability at 46 degrees C and completely reconstituted oxidase activity of heat-treated cytosol. These studies indicate that mild heating of either intact neutrophils or normal neutrophil cytosol results in a selective inactivation of p67-phox, providing a model oxidase system for the extremely rare p67-phox-deficient form of CGD.

Amino Acid Sequence↗
PubMed1992

Molecular basis of the autosomal recessive forms of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited group of disorders in which phagocytic leukocytes (neutrophils, eosinophils, monocytes, and macrophages) fail to undergo a respiratory burst when stimulated. The products of the respiratory burst, which include superoxide and hypochlorous acid, play a critical role in killing pathogenic bacteria, fungi, and parasites. As a result of the failure to activate the respiratory burst in their phagocytes, most CGD patients suffer from severe recurrent infections. While all CGD patients share this severe defect, there is substantial heterogeneity in the molecular mechanisms responsible. The enzyme that catalyzes the respiratory burst, NADPH oxidase, has been extensively characterized and found to consist of at least four subunits: gp91-phox and p22-phox (the two subunits of a low potential cytochrome b that is the terminal electron carrier of the oxidase) as well as p47-phox and p67-phox (two cytosolic oxidase components). CGD is caused by a defect in any one of these four components, thus explaining the previously confusing genetic heterogeneity of this disorder. In approximately thirty reported cases, the underlying mutations involving these oxidase components have been identified. The current understanding of the molecular basis of CGD is reviewed in the context of a recently completed Phase III clinical trial establishing the efficacy of recombinant human interferon gamma in the treatment of CGD.

Granulomatous Disease, Chronic↗
PubMed1992

Chronic granulomatous disease: diagnosis and classification at the molecular level.

Chronic granulomatous disease (CGD) is caused by the failure of phagocytes to produce microbicidal derivatives of molecular oxygen, such as hydrogen peroxide. It is one of the best characterized of the phagocyte disorders and represents an important consideration in the differential diagnosis of recurrent infections. The clinical, biochemical, and molecular genetic aspects of CGD are reviewed in this context in this article.

Amino Acid Sequence↗
PubMed1991

Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a congenital disorder in which phagocytes cannot generate superoxide (O2-) and other microbial oxidants due to mutations in any one of four components of the O2(-)-generating complex, NADPH oxidase. We report here a female CGD patient in whom a missense mutation in one of these components, the p22-phox subunit of the neutrophil membrane cytochrome b [where phox indicates phagocyte oxidase (used to designate protein components of the phagocyte NADPH oxidase)] results in a nonfunctional oxidase and failure of neutrophils to produce O2- in response to phorbol 12-myristrate 13-acetate. Cytochrome b in the patient's neutrophils was normal in appearance and abundance as determined by visible spectroscopy and by immunoblots of the gp91 and p22 subunits. However, the neutrophil plasma membranes were devoid of activity in the cell-free oxidase activation system, whereas the cytosol functioned normally. We postulated that the patient was homozygous for a mutation in p22 that results in the synthesis of normal levels of a nonfunctional cytochrome b. A single-base substitution (C----A) was found in the patient's mononuclear cell p22-phox cDNA that predicts a nonconservative Pro----Gln substitution at residue 156. The same mutation was also identified in all clones sequenced from patient genomic DNA, demonstrating homozygosity for the mutant allele. An antipeptide antibody against p22 residues 153-164 was found to bind only to permeabilized neutrophils, indicating that the mutation occurs in a cytoplasmic domain. These studies establish that this domain of p22-phox is cytoplasmic and that mutations in this region can have profound effects on cytochrome b function.

Adult↗
PubMed1991

Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2.

A major action of the microbicidal system of human neutrophils is the formation of superoxide anion (O2-) by a multicomponent oxidase that transfers electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen. The mechanism of assembly and activation of the oxidase from its cytosolic and membrane-bound components is unknown, but may require the activity of a guanosine 5'-triphosphate (GTP)-binding component. A cytosolic GTP-binding protein (Gox) that regulates the NADPH oxidase of neutrophils was identified. Gox was purified and shown to augment the rate of O2- production in a cell-free oxidase activation system. Sequence analysis of peptide fragments from Gox identified it as Rac 2, a member of the Ras superfamily of GTP-binding proteins. Antibody to a peptide derived from the COOH-terminus of Rac 2 inhibited O2- generation in a concentration-dependent manner. These results suggest that Rac 2 is a regulatory component of the human neutrophil NADPH oxidase, and provide new insights into the mechanism by which this oxygen radical-generating system is regulated.

Amino Acid Sequence↗
PubMed1991

Staurosporine inhibits the soluble and membrane-bound protein tyrosine kinases of human neutrophils.

Superoxide production by neutrophils triggered with a chemotactic peptide or a phorbol ester is inhibited by the protein kinase antagonists staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). We evaluated the effects of these antagonists on the protein tyrosine kinases and protein kinase C activities of neutrophils. Staurosporine completely inhibited all of these enzymes, whereas 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine was only substantially effective against protein kinase C. Thus, if a protein tyrosine kinase is involved in superoxide production, it is likely to function with a second kinase sensitive to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Identification of the NADPH-binding subunit of the respiratory burst oxidase.

The respiratory burst oxidase is a multicomponent membrane-bound enzyme that uses NADPH to reduce oxygen to O2-. When oxidase-containing membranes from activated neutrophils are treated with 0.3 M KCl, the NADPH-binding component of the oxidase elutes from the membranes in an active form. Treatment of this eluate with [32P]NADPH dialdehyde labels an approximately 32-kDa protein that is absent from eluates obtained from normal resting membranes or from resting or activated membranes from patients with one form of chronic granulomatous disease. We propose that this approximately 32-kDa protein is the NADPH-binding component of the oxidase.

Cell Membrane↗
PubMed1991

Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558.

The resonance Raman spectra of neutrophil cytochrome b558 obtained upon Soret excitation indicate that the heme is low spin six-coordinate in both ferric and ferrous oxidation states; comparison with the spectra of bis-imidazole hemin suggests imidazole or imidazolate axial ligation. Minor bands attributable to vibrational motions of ring-conjugated vinyl substituents were also observed, consistent with a heme assignment of protoporphyrin IX. The spectra of deoxycholate-solubilized cytochrome b558 were indistinguishable from neutrophil plasma membranes or specific granules, as were spectra from unstimulated and phorbol myristate acetate-stimulated cells, indicating that the hemes are structurally identical in various subcellular environments and cellular physiological states. However, structural complexity was suggested by biphasic ferric-ferrous photoreduction under 413-nm illumination and the absence of an EPR spectrum for the ferric heme under conditions where simple bis-imidazole heme-containing cytochromes are expected to give detectable signals. Midpoint reduction potentials and resonance Raman spectra of the soluble cytochrome b558 from an individual with cytochrome b558 positive (type IA.2) chronic granulomatous disease were nearly identical to normal oxidase, with the exception that the deficient oxidase did not undergo heme photoreduction. Possible structural models are discussed in relation to other physical properties (ligand binding, thermodynamic potentials) exhibited by the cytochrome.

Cell Membrane↗
PubMed1991

Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558.

Two of the cytosolic NADPH oxidase components, p47-phox and p67-phox, translocate to the plasma membrane in normal neutrophils stimulated with phorbol myristate acetate (PMA). We have now studied the translocation process in neutrophils of patients with chronic granulomatous disease (CGD), an inherited syndrome in which the oxidase system fails to produce superoxide due to lesions affecting any one of its four known components: the gp91-phox and p22-phox subunits of cytochrome b558 (the membrane-bound terminal electron transporter of the oxidase), p47-phox, and p67-phox. In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b558. In one patient with a rare X-linked form of CGD caused by a Pro----His substitution in gp91-phox, but whose neutrophils have normal levels of this mutant cytochrome b558, translocation was normal. In two patients with p47-phox deficiency, p67-phox failed to translocate, whereas p47-phox was detected in the particulate fraction of PMA-stimulated neutrophils from two patients deficient in p67-phox. Our data suggest that cytochrome b558 or a closely linked factor provides an essential membrane docking site for the cytosolic oxidase components and that it is p47-phox that mediates the assembly of these components on the membrane.

Biological Transport↗
PubMed1991

Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.

Resting and phorbol-activated human neutrophils were separated by treatment with Triton X-100 into detergent-extractable and cytoskeleton fractions. Respiratory burst oxidase activity was restricted entirely to the cytoskeleton. The cytoskeleton also contained approximately 15% of the neutrophil cytochrome b558, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67phox. In contrast, the components of the oxidase-associated phosphoprotein family p47phox were found almost exclusively in the detergent extract, suggesting that p47phox is needed for oxidase activation but not for O2- production by the activated oxidase. Activation of the oxidase had no apparent effect on the distribution of any of these species between the cytoskeleton and the detergent extract. Our results support earlier studies implying that the cytoskeleton participates in an important way in regulating the activity of the O2(-)-forming respiratory burst oxidase of neutrophils.

Blotting, Western↗