Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.

The superoxide-forming respiratory burst oxidase of human neutrophils is composed of membrane-associated catalytic components and cytosolic constituents required for oxidase activation. This study concerns the hypothesis that cytosolic oxidase components translocate to a membrane fraction when neutrophils are stimulated and the oxidase is activated. A polyclonal antiserum that recognizes two discrete cytosolic oxidase components of 47 and 67 kD was used to probe transfer blots of electrophoresed membrane and cytosol fractions of resting and stimulated neutrophils. In contrast to their strictly cytosolic localization in unstimulated cells, both proteins were detected in membrane fractions of neutrophils activated by phorbol esters and other stimuli. This translocation event was a function of stimulus concentration as well as time and temperature of exposure to the stimulus. It was inhibited by concentrations of N-ethylmaleimide that blocked superoxide formation but was unaffected by 2-deoxyglucose. There was a correlation between translocation of the cytosolic proteins and activation of the oxidase as determined by superoxide formation. Quantitative analyses suggested that approximately 10% of total cellular p47 and p67 became membrane-associated during phorbol ester activation of the oxidase. Analysis of Percoll density gradient fractions indicated that the target membrane for translocation of both proteins was the plasma membrane rather than membranes of either specific or azurophilic granules. In the cell-free oxidase system arachidonate-dependent but membrane-independent precipitation of the cytosolic oxidase proteins was demonstrated. The data show that activation of the respiratory burst oxidase in stimulated human neutrophils is closely associated with translocation of the 47- and 67-kD cytosolic oxidase components to the plasma membrane. We suggest that this translocation event is important in oxidase activation.

Inhibition by carboxamides and sulfoxides of liver alcohol dehydrogenase and ethanol metabolism.

Sulfoxides and amides were tested as inhibitors of liver alcohol dehydrogenase and ethanol metabolism in rats. With both series of compounds, increasing the hydrophobicity resulted in better inhibition, and introduction of polar groups reduced inhibition. Of the cyclic sulfoxides, tetramethylene sulfoxide was the best inhibitor as compared to the tri- and pentamethylene analogue and other compounds, and it may be a transition-state analogue. The most promising compounds, tetramethylene sulfoxide and isovaleramide, were essentially uncompetitive inhibitors of purified horse and rat liver alcohol dehydrogenases with respect to ethanol as substrate. These compounds also were uncompetitive inhibitors in vivo, which is advantageous since the inhibition is not overcome at higher concentrations of ethanol, as it is with competitive inhibitors, such as pyrazole. The uncompetitive inhibition constants for tetramethylene sulfoxide and isovaleramide for rat liver alcohol dehydrogenase were 200 and 20 microM, respectively, in vitro, whereas in vivo the values were 340 and 180 mumol/kg. The differences in the values may be due to metabolism or distribution of the compounds. Further studies will be required to determine if isovaleramide or tetramethylene sulfoxide is suitable for therapeutic purposes.

Inhibition of liver alcohol dehydrogenase and ethanol metabolism by 3-substituted thiolane 1-oxides.

3-Substituted thiolane 1-oxides (methyl, n-butyl, n-hexyl, and phenyl) were prepared and tested as inhibitors of horse, monkey, and rat liver alcohol dehydrogenases and of ethanol metabolism in rats. These compounds inhibit alcohol oxidation in an uncompetitive manner with respect to ethanol as a varied substrate. Lengthening the alkyl substituent increased the inhibitory potency because of tighter binding in the hydrophobic substrate binding pocket of the alcohol dehydrogenases. Thus, the 3-hexyl derivative was the most potent inhibitor of the purified rat liver alcohol dehydrogenase, with a Kii value of 0.13 microM. The 3-butyl derivative was the best inhibitor of ethanol metabolism in rats, with a Kii value of 11 mumol/kg. The acute toxicity in mice of the butyl derivative was 1.4 mmol/kg. Since high concentrations of alcohol do not prevent the inhibitory effects of these compounds, they may be particularly useful for preventing poisoning by methanol or ethylene glycol.

Oxidative inactivation of Actinobacillus actinomycetemcomitans leukotoxin by the neutrophil myeloperoxidase system.

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils.

Small molecular weight secretory factors from Pseudomonas aeruginosa have opposite effects on IL-8 and RANTES expression by human airway epithelial cells.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes both an acute lung disease in patients with hospital-acquired pneumonia and a chronic lung disease in individuals with cystic fibrosis. Many of the pathophysiologic effects of P. aeruginosa infection are due to factors secreted by the bacterium. Conditioned media from cultures of P. aeruginosa increased interleukin-8 expression and decreased regulated on activation, normal T cells expressed and secreted (RANTES) expression by human airway epithelial cells. Both of these activities were present in heat-treated, protease-treated, small molecular weight fractions. The activities were not inhibited by polymyxin B and were not extracted into ethyl acetate, suggesting that they were not due to endotoxin or autoinducer. Conversely, results from chloroform extractions and studies with a phenazine-minus mutant suggested that the blue pigment pyocyanin contributes to these activities when present. In addition to the effects of small molecular weight factors on cytokine expression, proteases in bacterial-conditioned media further decreased levels of RANTES. By altering expression, release, and/or activity of inflammatory cytokines, secretory factors from P. aeruginosa could disrupt the delicate balance that constitutes the immune response to bacterial infection and thus could contribute to the lung damage that occurs in P. aeruginosa-infected airways.

Functional domain in an arginine-rich carboxyl-terminal region of p47phox.

Activation of the neutrophil respiratory burst oxidase involves phosphorylation-dependent translocation of the cytosolic proteins p47phox and p67phox to the plasma membrane, a process in intact cells that is inhibited by staurosporine. We now report that in a cell-free oxidase system, staurosporine and protein kinase C pseudosubstrate PKC(19-36) both inhibited p47phox phosphorylation but had no effect on superoxide generation. In contrast, p47phox phosphorylation, translocation, and superoxide generation were inhibited by a peptide, p47phox(323-332) (AYRRNSVRFL), based on a putative serine phosphorylation domain. This effect was specific for the 323-332 peptide, as it was not observed with two peptides based on other p47phox phosphorylation domains. All three peptides served as substrates for phosphorylation, but the extent of peptide phosphorylation did not correlate with inhibition of oxidase function. p47phox(325-330), which represents the serine phosphorylation motif of p47phox(323-332), did not inhibit translocation or superoxide production despite its ability to block phosphorylation of p47phox. These data indicate the presence of functionally important sites within the p47phox(323-332) peptide. Although serine 328 is in a consensus phosphorylation motif, the lack of correlation in the cell-free system between p47phox phosphorylation and either protein translocation or superoxide formation suggests that a phosphorylation-independent function resides in the 323-332 segment of p47phox.

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system.

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase.

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.

Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components.

Activated human polymorphonuclear neutrophils (PMNs) convert molecular oxygen into superoxide anion, a process known as the respiratory burst, through the activity of a latent multicomponent NADPH-dependent oxidase. Components of this respiratory burst oxidase include the membrane-bound cytochrome b558 and the cytosolic factors p47-phox and p67-phox. We initiated these studies based on three observations: 1) that stimulation of PMN oxidase activity is associated with translocation of the cytosolic oxidase components to the plasma membrane; 2) that p47-phox is phosphorylated during PMN activation and that there is a sequential relationship between phosphorylation of p47-phox in the cytosol and appearance of the phosphoprotein in the membran; and 3) that the predicted amino acid sequences of p47-phox and of p67-phox contain regions of homology to the SH3 or A domain of the src family of tyrosine kinases, a region found in a variety of proteins which interact with the cytoskeleton or the subplasmalemmal cytoskeleton. Thus the purpose of our studies was to examine the role of protein kinase C (PKC)-dependent phosphorylation in the stimulus-induced association of p47-phox and p67-phox with the plasma membrane and the cytoskeleton. Using the PKC activator phorbol myristate acetate (PMA) as the agonist, we found that activation of the respiratory burst oxidase was associated with translocation of cytosolic p47-phox and p67-phox to the plasma membrane as well as redistribution of p47-phox to the Triton-insoluble cytoskeleton. Furthermore, the PKC inhibitor staurosporine inhibited phosphorylation of p47-phox, interrupted the redistribution of cytosolic oxidase factors, and blocked PMA-induced generation of superoxide anion. Taken together these results indicate that PKC-dependent phosphorylation of p47-phox correlates with association of p47-phox with the cytoskeleton and with translocation of p47-phox and p67-phox to the plasma membrane, with the ensuing assembly of an active superoxide-generating NADPH-dependent oxidase.

Characterization of alcohol dehydrogenase from cultured rat hepatoma (HTC) cells.

The highly active alcohol dehydrogenase (EC 1.1.1.1) in rat hepatic tumor cells (HTC) was purified 120-fold by chromatography on DEAE-Sepharose and AMP-Agarose to yield an enzyme with a specific activity of 88 mumole/min/mg protein, assayed with 1.7 mM NAD+ and 0.55 M ethanol at pH 9 and 30 degrees C. (By comparison, purified, normal rat liver enzyme has an activity of about 1 unit/mg.) Based on its physical and kinetic properties, we conclude that the HTC isozyme is the same as the enzyme from rat stomach and another rat hepatoma (Cederbaum AI, Pietruszko R, Hempel J, Becker FF, and Rubin E (1975) Arch Biochem Biophys 171:348-360). The kinetics of the HTC enzyme are consistent with the Ordered Bi Bi mechanism. The kinetic constants are generally much larger for the HTC enzyme than for the normal rat liver enzyme. The Michaelis constants for ethanol and acetaldehyde (Kb = 1100 mM, Kp = 260 mM) are 1000-fold larger, and the constants for NADH are 10 to 50-fold larger. Although the HTC enzyme has low catalytic efficiency (V/Kb) on ethanol, it has much better activity on longer chain alcohols, but no activity on cyclohexanol. The pH dependence of V/Kb with ethanol is unusual in that it appears to be a linear function of pH, increasing with a slope of 0.56. Thus, the active sites of the liver and HTC enzymes may be different, although the HTC enzyme is inactivated by bromoacetate and bipyridine as is found for the liver enzyme. The HTC (stomach) enzyme may function to oxidize high concentrations of ingested ethanol or longer chain alcohols.

omega-haloalkyl esters of 5'-adenosine monophosphate as potential active-site-directed reagents for dehydrogenases.

A series of esters of adenosine 5'-monophosphate with ethyl, propyl, or hexyl moieties substituted at the omega-position with chlorine or bromine were prepared. The compounds were competitive inhibitors of horse liver alcohol dehydrogenase with respect to coenzyme, NAD+, and had inhibition (dissociation) constants in the range of 40 to 260 microM at pH 8.0, 25 degrees C. The bromoalkyl esters were designed to be active-site-directed inactivators and were chemically reactive as tested with the model compound 4-(p-nitrobenzyl)pyridine. Yeast alcohol dehydrogenase was inactivated by the bromohexyl analog by an active-site-directed mechanism, with a Ki = 1.5 mM and a pseudo-bimolecular rate constant of 0.03 M-1 S-1, which is 150 times larger than the bimolecular rate constant for inactivation by 2-bromoethanol. However, the rates of inactivation of other dehydrogenases treated with 10 mM concentrations of these compounds were generally slower than with the simpler reagent, 2-bromoethanol. Thus, the reactive functional group attached to the AMP moiety may not be properly oriented for affinity labeling of these dehydrogenases. The bromoalkyl esters may be useful for inactivating other enzymes.

Calreticulin biosynthesis and processing in human myeloid cells: demonstration of signal peptide cleavage and N-glycosylation.

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.