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B M Babior

Publications and source records attributed to B M Babior.

At least 55 records · Page 3Linked to original sources
PubMed1995

Purification and characterization of a C5a-inactivating enzyme from human peritoneal fluid.

Earlier work has suggested that familial Mediterranean fever, an inherited disorder characterized by sporadic episodes of inflammation involving the pleural and peritoneal cavities and the joints, is caused by the lack of a C5a inactivator normally found in serosal fluid. We have purified this inactivator from ascites fluid and obtained a protein of molecular weight 53 to 56 kD with a specific activity 10,000-fold greater than the crude material. On Western blot, an inhibitory antibody recognized a single antigenic species at the same molecular weight. The enzyme had no activity against denatured bovine serum albumin. With recombinant C5a as substrate, the Km and Vm were 3.4 mumol/L and 52 nmol C5a/min/mg protein, respectively.

Ascitic Fluid↗
PubMed1995

Cell-free activation of the respiratory burst oxidase by protein kinase C.

In intact neutrophils, phorbol ester treatment activates the respiratory burst oxidase, the enzyme responsible for O2-production by phagocytes. This effect is thought to be dependent on protein kinase C and on the phosphorylation of p47phox. In this paper, we report that protein kinase C activates the respiratory burst oxidase in a cell-free system consisting of isolated neutrophil cytosol and membrane. Oxidase activation required a highly active protein kinase C, recombinant p47phow and ATP, and was inhibited by the protein kinase C inhibitors H-7 and GF-109203X. PERIl depletion of cytosolic ATP by dialysis reduced oxidase activation by over 50% In contrast, neither protein kinase C inhibitors nor ATP depletion affected oxidase activation by SDS. These findings strongly suggest that in the cell-free system, the oxidase can be activated by the phosphorylation of p47phox.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit.

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

Amino Acid Sequence↗
PubMed1995

The respiratory burst oxidase.

The respiratory burst oxidase catalyzes the production of O2- by activated phagocytes and B lymphocytes. Activation is accomplished by any number of signal transduction pathways, and involves protein kinase C, MAP kinase, or both, and perhaps lipid-mediated pathways. Failure of O2- production is characteristic of chronic granulomatous disease, an inherited disorder of phagocyte function. A number of new mutations responsible for chronic granulomatous disease have been reported. O2- production is also altered in other diseases, most notably certain hematologic malignancies.

Enzyme Activation↗
PubMed1995

The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O2- at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303 and S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47phox after transfection with plasmids expressing various S-->A mutants of p47phox. The mutant containing S-->A mutations involving all serines between S303 and S379 [S(303-379)A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47 phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47phox, and (b) p47phox S379A failed to translocate to the membrane, and was as functionless as p47phox S(303-379)A; other single S-->A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.

Amino Acid Sequence↗
PubMed1994

The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases.

The respiratory burst oxidase catalyzes the production of O2.- from oxygen and NADPH. It is dormant in resting cells but becomes active when the cells are stimulated. Activation is accompanied by the phosphorylation of multiple serines in the cytosolic oxidase component p47phox, which moves from cytosol to the membrane during oxidase activation. Using immunopurified p47phox isolated from 32Pi-loaded neutrophils activated with phorbol myristate acetate, we showed that all the 32P was in the C-terminal CNBr fragment of the protein, and that in that fragment, Ser-303, Ser-304, Ser-320, Ser-328, Ser-345, and Ser-348 and at least one of the three serines, Ser-359, Ser-370, and Ser-379, were phosphorylated, while Ser-282, Ser-287, Ser-381, and Ser-388 were not. Of the phosphorylated serines, Ser-303, Ser-304, Ser-320, and Ser-328 are located in protein kinase C substrate sequences. Ser-345 and Ser-348, however, are located in sequences recognized by mitogen-activated protein (MAP) kinase (-PXSP-). This finding suggests that MAP kinase or a related proline-directed kinase may participate in the regulation of O2.- production by activated neutrophils. The tryptic peptide map of p47phox phosphopeptides from neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine closely resembled that of p47phox phosphopeptides from phorbol-activated cells, suggesting that the same serines were phosphorylated in response to each agent.

Amino Acid Sequence↗
PubMed1994

Isolation of a complex of respiratory burst oxidase components from resting neutrophil cytosol.

The respiratory burst oxidase of neutrophils is a multicomponent enzyme, dormant in resting cells, that catalyzes the reduction of oxygen to O2- at the expense of NADPH. In the resting neutrophil, some of the components of the oxidase, including proteins p47phox and p67phox, are in the cytosol, while the rest are in a fraction that usually copurifies with plasma membrane. Recent evidence has suggested that at least some of the cytosolic oxidase components exist as a complex. We have now purified such a complex from the cytoplasm of resting neutrophils using an affinity column prepared with an antibody that recognizes the C-terminal decapeptide of p47phox. Immunoblotting showed that the complex contained both p47phox and p67phox. When supplemented with recombinant p67phox, the complex displayed considerable activity in a cell-free oxidase-activating system, and even without added p67phox, the complex could more than double O2- production in an oxidase-activating system supplemented with suboptimal amounts of cytosol. Isolation of the complex was blocked by preincubating the affinity column with CFSTKRKLASAV, the peptide against which the antibody was raised. On gel filtration, the complex migrated with a molecular weight of 240-300K, similar to that observed with whole neutrophil cytosol. The p47phox/p67phox ratio in the gel-filtered complex was approximately 1 to 1. These results indicate that in resting neutrophil cytosol, p47phox and p67phox exist as a complex.

Amino Acid Sequence↗
PubMed1994

Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation.

The respiratory burst oxidase is responsible for O2- production in stimulated neutrophils and B lymphocytes. Components of this oxidase include cytochrome b558, a membrane-bound flavohemoprotein; the cytosolic polypeptides p47phox and p67phox; and one or more small G proteins including Rac1, Rac2, and/or Rap1A. We found that when normal neutrophils were activated, small percentages of each of the cytosolic proteins p47phox, p67phox, and Rac2 were transferred to the membrane cytoskeleton. However, Rac2 was not transferred to the membrane during activation of p47phox-deficient neutrophils. In normal cells, some p47phox also became associated with the non-cytoskeletal portion of the plasma membrane, but p67phox, Rac2, and O(2-)-forming activity were restricted to the cytoskeleton. Neutrophil activation also causes the phosphorylation of multiple serines in p47phox. The most heavily phosphorylated forms of p47phox were found solely in the membrane cytoskeleton. These results suggest that 1) the membrane cytoskeleton participates in respiratory burst oxidase activation, 2) the fully phosphorylated p47phox is located in the active oxidase, which resides in the membrane cytoskeleton, and 3) Rac2 acts like a dedicated component of the respiratory burst oxidase.

Actins↗
PubMed1994

Reperfusion injury induces apoptosis in rabbit cardiomyocytes.

The most effective way to limit myocardial ischemic necrosis is reperfusion, but reperfusion itself may result in tissue injury, which has been difficult to separate from ischemic injury. This report identifies elements of apoptosis (programmed cell death) in myocytes as a response to reperfusion but not ischemia. The hallmark of apoptosis, nucleosomal ladders of DNA fragments (approximately 200 base pairs), was detected in ischemic/reperfused rabbit myocardial tissue but not in normal or ischemic-only rabbit hearts. Granulocytopenia did not prevent nucleosomal DNA cleavage. In situ nick end labeling demonstrated DNA fragmentation predominantly in myocytes. The pattern of nuclear chromatin condensation was distinctly different in reperfused than in persistently ischemic tissue by transmission electron microscopy. Apoptosis may be a specific feature of reperfusion injury in cardiac myocytes, leading to late cell death.

Animals↗
PubMed1994

Activation of the respiratory burst oxidase.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen by NADPH to form O2-, the precursor of a group of reactive oxidants that are employed by phagocytes as microbicidal agents. The enzyme is active in stimulated cells but dominant in resting cells. It molecular weight guanine nucleotide-binding protein. The components p22phox and gp91phox from cytochrome b558, a flavohemoprotein that resides in the cortical cytoskeleton and in the membranes of the specific granules. The other components are found in the cytosol of resting cells, but migrate to the cortical cytoskeleton when the neutrophils are activated, where they assemble the active oxidase. Migration to the cortical cytoskeleton is caused in part by the appearance of a membrane binding site on one or more of the cytosolic subunits, possibly due to the phosphorylation of p47phox that takes place during cell activation.

B-Lymphocytes↗
PubMed1993

Inactivation of interleukin-8 by the C5a-inactivating protease from serosal fluid.

The complement fragment C5a and the cytokine interleukin-8 (IL-8) are proinflammatory peptides with potent chemotactic activity toward neutrophils. We have previously shown that C5a can be inactivated by a protease that is found in normal synovial and peritoneal fluids but is absent from serosal fluids obtained from patients with familial Mediterranean fever (FMF). We report here that serosal fluids can also eliminate the chemotactic activity of IL-8. The agent responsible for IL-8 elimination appears to be the C5a-inactivating protease, because the pure protease can inactivate IL-8, inactivation of IL-8 by normal peritoneal fluid is partly prevented by an antibody raised against the purified C5a-inactivating protease, and IL-8 is not inactivated by peritoneal fluids from patients with FMF. The ability of this protease to inactivate both, early (C5a) and late (IL-8) inflammatory mediators identifies it as a potentially significant regulator of inflammation.

Ascitic Fluid↗
PubMed1993

Effects of diacylglycerol on the activation and kinetics of the respiratory burst oxidase in a cell-free system from human neutrophils: evidence that diacylglycerol may regulate nucleotide uptake by a GTP-binding protein.

The respiratory burst oxidase from human neutrophils is a membrane-associated enzyme that catalyzes the reduction of oxygen to O2- at the expense of NADPH. The oxidase is dormant in resting neutrophils, but comes to life when the cells are exposed to certain activating agents. Activation requires GTP or a GTP analog and is associated with the transfer of cytosolic oxidase polypeptides to the plasma membrane. Treatment of resting neutrophil membranes with phospholipase C caused an eightfold rise in their diacylglycerol content, increased the sodium dodecyl sulfate-mediated transfer of cytosolic polypeptides to the membrane, and enhanced O2- production by the membranes after treatment with cytosol and sodium dodecyl sulfate. Use of phospholipase C-treated membranes in the cell-free system caused only a minor change in the Km and Vm for NADPH as compared with the same system containing untreated membranes, but caused the Km for the nonhydrolyzable GTP analog GTP gamma S to fall from 200 to 34 nM. Similar kinetic changes were observed with membranes treated with dioctanoylglycerol. These findings are consistent with the idea that the activity of a G protein can be regulated by diacylglycerol.

Cell-Free System↗
PubMed1992

The reduction of cytochrome b558 and the activity of the respiratory burst oxidase from human neutrophils.

It is known that in respiratory burst oxidase preparations engaged in O2- production, cytochrome b558, a characteristic oxidase component, is partly reduced. This result has been interpreted in terms of a mechanism in which cytochrome b558 functions as an electron-carrying component of the respiratory burst oxidase, its level of reduction reflecting a steady-state partitioning of the cytochrome between reduced and oxidized forms as it ferries electrons from NADPH to oxygen. Kinetic arguments based on this interpretation have supported the proposal that the cytochrome is reduced at a rate sufficient to account for the rate of O2- production by activated neutrophils. We have confirmed the partial reduction of cytochrome b558 in neutrophil cytoplasts and in oxidase preparations exposed to NADPH, but have found that the reduction of the cytochrome bears no apparent relation to the activity of the oxidase, and can occur when NADPH is added to neutrophil membrane preparations that are unable to manufacture O2-. We therefore conclude that the NADPH-dependent reduction of cytochrome b558 seen in these preparations is unlikely to be a reflection of a catalysis-related steady state and that inferences drawn from such observations regarding the kinetic competence of the cytochrome may need to be reconsidered.

Alkaline Phosphatase↗
PubMed1992

O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA.

The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every chronic granulomatous disease patient studied to date, an abnormality has been found in a gene encoding one of four components of the respiratory burst oxidase: the membrane protein p22phox or gp91phox, or the cytosolic protein p47phox or p67phox. We report here that O2- production was partly restored to phorbol 12-myristate 13-acetate-stimulated Epstein-Barr virus-transformed B lymphocytes from a patient with p47phox-deficient chronic granulomatous disease by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. No detectable O2- was produced by untransfected p47phox-deficient lymphocytes or by p47phox-deficient lymphocytes transfected with an antisense plasmid. The finding that O2- can be produced by p47phox-deficient B lymphocytes after the transfer of a p47phox cDNA into the deficient cells suggests that this system could be useful for studying the function of mutant p47phox proteins in whole cells.

Amino Acid Sequence↗
PubMed1992

The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol.

The respiratory burst oxidase is a multimeric enzyme responsible for O2- production by stimulated neutrophils and a few other cell types. In the resting neutrophil, the oxidase is dormant, and its subunits are distributed between the cytosol, in which they appear to exist in the form of a multisubunit complex, and the plasma membrane; but, when the neutrophil is activated, the cytosolic complex translocates to the membrane to assemble the active enzyme. Using a cell-free system in which oxidase activity was elicited with SDS, we examined the effects of GTP gamma S and dioctanoylglycerol (DiC8) on both the activation of O2- production and the transfer of the cytosolic oxidase components p47phox and p67phox to the plasma membrane. GTP (added as undialyzed cytosol) and GTP gamma S augmented the transfer of the oxidase components to the plasma membrane and was essential for the acquisition of O2- producing activity by the oxidase. DiC8 also supported the SDS-mediated transfer of oxidase components to the membrane, but O2- production did not take place unless GTP or GTP gamma S was present. In the presence of these nucleotides, however, DiC8 augmented both translocation and O2- production. We interpreted these results in terms of a mechanism in which 2 membrane-binding sites are created during the activation of the cytosolic complex, one for diacylglycerol and the other for a second site on the membrane. Development of the second membrane-binding site depends upon the action of a G protein and is essential for the expression of oxidase activity. The results further suggested that the priming of the respiratory burst oxidase in intact neutrophils might be due to an increase in membrane diacylglycerol concentration that occurs in response to the priming stimulus. Because of the increased diacylglycerol content, a larger than usual amount of active respiratory burst oxidase could be assembled on the primed plasma membrane when the neutrophil is fully activated.

Adult↗
PubMed1992

The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system.

Sodium dodecyl sulfate (SDS) treatment of a mixture of cytosol and plasma membranes from resting neutrophils resulted in the activation of the respiratory burst oxidase, a complicated enzyme that catalyzes the production of O2- from NADPH and oxygen. Activation was accompanied by translocation to the plasma membranes of the oxidase components p47phox and p67phox, which in resting cytosol were found in a M(r) approximately 240,000 complex. This translocation, which appeared to take place without a major change in the size of the cytosolic complex, did not occur if the membranes lacked cytochrome b558, and was inhibited by the peptide PRGV-HFIFNK, a sequence found near the carboxyl terminus of cytochrome b558 that was known from earlier work to inhibit O2- production by the cell-free system (Rotrosen, D., Kleinberg, M. E., Nunoi, H., Leto T., Gallin, J. I., and Malech H. L. (1990) J. Biol. Chem. 265, 8745-8750). Cytosols pretreated with the cross-linking agents 3,3'-dithiobis(sulfosuccinimidyl) propionate (DTSSP) (cleavable by 2-mercaptoethanol) and bis-(sulfosuccinimidyl) suberate (not cleavable by 2-mercaptoethanol) lost most of their ability to support O2- production in the cell-free system, and oxidase components from DTSSP-treated cytosol failed to translocate to the plasma membrane. When DTSSP-treated cytosols were incubated with 2-mercaptoethanol, however, both O2- production and translocation were partly restored, indicating that the functional impairment in DTSSP-treated cytosols was probably due at least in part to a restriction in the conformational mobility of the cross-linked peptide chains in the approximately 240,000 complex. These findings provide further support for the idea that the cytosolic components of the respiratory burst oxidase exist in the form of a approximately 240,000 complex, and suggest that the exposure of this complex to SDS induces a structural change that may or may not be associated with the loss of an inhibitory subunit too small to cause a detectable change in the size of the complex. This SDS-induced change allows translocation to take place by creating a membrane-binding site on the surface of the complex.

Adult↗
PubMed1992

The respiratory burst oxidase.

Sbarra and Karnovsky were the first to present evidence suggesting the presence in phagocytes of a special enzyme designed to generate reactive oxidants for purposes of host defense. In the years since their report appeared, a great deal has been learned about this enzyme, now known as the respiratory burst oxidase. It has been found to be a plasma membrane-bound heme- and flavin-containing enzyme, dormant in resting cells, that catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH: O2 + NADPH----O2- + NADP+ + H+ Its behavior in whole cells and its response to various activating stimuli have been described in detail, although important insights continue to emerge, as for example a very interesting new series of observations on differences in oxidase activation patterns between suspended and adherent cells. The enzyme has been shown by biochemical and genetic studies to consist of at least six components. In the resting cell, three of these components are in the cytosol and three in the plasma membrane, but when the cell passes from its resting to its activated state the cytosolic components are all transferred to the plasma membrane, presumably assembling the oxidase. Of the components initially bound to the membrane, two constitute cytochrome b558, a heme protein characteristic of the respiratory burst oxidase, and the third may represent an oxidase flavoprotein. With regard to the cytosolic components, one is a phosphoprotein and another is the NADPH-binding component, possibly a second oxidase flavoprotein. The nature of the third (p67phox) is a puzzle. Four of the six oxidase components have now been cloned and sequenced. These findings only scratch the surface, however, and many questions remain. How many oxidase components, for example, remain to be discovered, and how do they fit together to form the active enzyme? How is the route of activation of the oxidase integrated into the general signal transduction systems of the cell? How did the oxidase come to be? Could there be a widespread system that generates small amounts of O2- as an intercellular signaling molecule, as recent work is beginning to suggest, and did the ever-destructive respiratory burst oxidase arise from that innocuous system as the creation of some evolutionary Frankenstein--an oxidase from hell? Finally, will it be possible to develop drugs that specifically block the respiratory burst oxidase, and will such drugs prove to be clinically useful as anti-inflammatory agents?(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗