Inactivation of alpha 1-antiproteinase by hydroxyl radicals. The effect of uric acid

The elastase-inhibitory activity of alpha 1-antiproteinase is inactivated by hydroxyl radicals (.OH) generated by pulse radiolysis or by reaction of iron ions with H2O2 in the presence of superoxide or ascorbate. Uric acid did not protect alpha 1-antiproteinase against inactivation by .OH in pulse radiolysis experiments or in the superoxide/iron/H2O2 system, whereas it did in systems containing ascorbic acid. We propose that radicals formed by attack of .OH on uric acid are themselves able to inactivate alpha 1-antiproteinase, but that these uric acid radicals can be 'repaired' by ascorbic acid.     

The antioxidant action of ergothioneine.

Ergothioneine is a product of plant origin that accumulates in animal tissues. Its suggested ability to act as an antioxidant has been evaluated. Ergothioneine is a powerful scavenger of hydroxyl radicals (.OH) and an inhibitor of iron or copper ion-dependent generation of .OH from hydrogen peroxide (H2O2). It is also an inhibitor of copper ion-dependent oxidation of oxyhaemoglobin, and of arachidonic acid peroxidation promoted by mixtures of myoglobin (or haemoglobin) and H2O2. Ergothioneine is a powerful scavenger of hypochlorous acid, being able to protect alpha 1-antiproteinase against inactivation by this molecule. By contrast, it does not react rapidly with superoxide (O2-) or hydrogen peroxide (H2O2) and it does not inhibit microsomal lipid peroxidation in the presence of iron ions. Overall, our results show that ergothioneine at the concentrations present in vivo could act as an antioxidant.     

Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.     

Molecular cloning and sequence analysis of cDNAs coding for guinea pig alpha 1-antiproteinases S and F and contrapsin

The cDNAs encoding two isoforms, S (slow) and F (fast), of alpha 1-antiproteinase (also referred to as alpha 1-antitrypsin or alpha 1-proteinase inhibitor) as well as contrapsin were obtained by screening lambda gt11 cDNA library prepared fro inflamed guinea pig liver. The sequence analyses of these cDNAs and NH2-terminal peptides of the purified proteins revealed that both isoforms of alpha 1-antiproteinase consist of 405 amino acid residues including a signal peptide of 24 residues and that contrapsin consists of 410 amino acid residues with the same length of the signal peptide. Guinea pig contrapsin had 89, 88, 62, 42, and 41% homology to its own alpha 1-antiproteinases F and S, rat alpha 1-antiproteinase, mouse and rat contrapsins, respectively. This suggests that guinea pig contrapsin is not orthologous to mouse and rat contrapsins and that it developed from a much later duplication of alpha 1-antiproteinase gene after the guinea pig had diverged from the murine lineage. The available data suggest that the reactive site region of alpha 1-antiproteinase can be categorized into orthodox and unorthodox types: the former has P3-P'3 consensus sequence of Xaa-Pro-Met-Ser-Xaa-Pro, where Xaa is Leu, Ile, Val, or Met, while the latter, which occurs in species having multiple alpha 1-antiproteinase isoforms, has the sequence whose P1 Met has changed to other amino acids. Thus, the reactive site region of the orthodox type, which occurs in all seven mammals examined to date, is highly conserved. This is in marked contrast to the fact that the same region is hypervariable among the paralogous proteins belonging to the serpin superfamily.     

The antioxidant action of taurine, hypotaurine and their metabolic precursors.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.     

Calcium paradox of aldosteronism and the role of the parathyroid glands

The hypercalciuria and hypermagnesuria that accompany aldosteronism contribute to a fall in plasma ionized extracellular Ca2+ and Mg2+ concentrations ([Ca2+]o and [Mg2+]o). Despite these losses and the decline in extracellular levels of these cations, total intracellular and cytosolic free Ca2+ concentration ([Ca2+]i) is increased and oxidative stress is induced. This involves diverse tissues, including peripheral blood mononuclear cells (PBMC) and plasma. The accompanying elevation in plasma parathyroid hormone (PTH) and reduction in bone mineral density caused by aldosterone (Aldo)-1% NaCl treatment (AldoST) led us to hypothesize that Ca2+ loading and altered redox state are due to secondary hyperparathyroidism (SHPT). Therefore, we studied the effects of total parathyroidectomy (PTx). In rats receiving AldoST, without or with a Ca2+-supplemented diet and/or PTx, we monitored urinary Ca2+ and Mg2+ excretion; plasma [Ca2+]o, [Mg2+]o, and PTH; PBMC [Ca2+]i and H2O2 production; plasma alpha1-antiproteinase activity; total Ca2+ and Mg2+ in bone, myocardium, and rectus femoris; and gp91(phox) labeling in the heart. We found that 1) the hypercalciuria and hypermagnesuria and decline (P < 0.05) in plasma [Ca2+]o and [Mg2+]o that occur with AldoST were not altered by the Ca2+-supplemented diet alone or with PTx; 2) the rise (P < 0.05) in plasma PTH with AldoST, with or without the Ca2+-supplemented diet, was prevented by PTx; 3) increased (P < 0.05) PBMC [Ca2+]i and H2O2 production, increased total Ca2+ in heart and skeletal muscle, and fall in bone Ca2+ and Mg2+ and plasma alpha1-antiproteinase activity with AldoST were abrogated (P < 0.05) by PTx; and 4) gp91(phox) activation in right and left ventricles at 4 wk of AldoST was attenuated by PTx. AldoST is accompanied by SHPT, with parathyroid gland-derived calcitropic hormones being responsible for Ca2+ overload in diverse tissues and induction of oxidative stress. SHPT plays a permissive role in the proinflammatory vascular phenotype.     

Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration

To investigate the mechanisms of H2O2 adaptation in mammalian cells, we exposed human U937 leukemia cells to 0.05 mM H2O2. This treatment significantly suppressed cell death and DNA fragmentation induced by a subsequent challenge with 1 mM H2O2. A more dramatic protection was observed when cells were pretreated with 0.25 mM H2O2. Pretreatment with either 0.05 or 0.25 mM H2O2 also imparted cells with a survival advantage against serum withdrawal and C2-ceramide treatment. H2O2 was found to be a mediator of cell death induced by serum withdrawal, but not by the addition of C2-ceramide. Interestingly, 0.25 mM H2O2 greatly induced glutathione peroxidase, a H2O2-consuming enzyme, whereas 0.05 mM H2O2 did not. Consistent with observation, pretreatment with 0.25 mM H2O2 resulted in a great reduction of cellular oxidant levels as determined by 2'7'-dichlorofluorescein fluorescence, and it also prevented elevation of oxidant levels upon subsequent challenge with 1 mM H2O2 or with serum withdrawal. These effects were not observed in cells pretreated with 0.05 mM H2O2. The sum of the data indicated that H2O2 suppresses cell death by two different mechanisms depending on its concentration: Relatively high concentrations enhance cellular antioxidant capacity, and lower concentrations block the lethal action of H2O2.     

NF-kappa B mediates the adaptation of human U937 cells to hydrogen peroxide

Low doses of oxidative stress can induce cellular resistance to subsequent higher doses of the same stress. By using human U937 leukemia cells, we previously demonstrated that H(2)O(2) can induce such an adaptive response without elevating the cellular capacity to degrade H(2)O(2), and were able to confer the cells a cross-resistance to an H(2)O(2)-independent lethal stimulus, C(2)-ceramide. In this study, it was found that the adaptation is accompanied by the translocation of cytoplasmic NF-kappa B to the nuclei. This event was promoted or abolished when either IKK alpha or a dominant negative mutant of I kappa B, respectively, was overexpressed. The overexpression of IKK alpha also resulted in the suppression of H(2)O(2)-induced cell death and DNA fragmentation, whereas these events were accelerated by the expression of the I kappa B mutant. The protective effect of IKK alpha was accompanied neither by an elevation of protein levels of various antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, nor by an increase in the cellular capacity to consume H(2)O(2). Moreover, the overexpression of IKK alpha resulted in an enhancement of H(2)O(2)-induced resistance to C(2)-ceramide. The overall data suggest that NF-kappa B mediates the H(2)O(2) adaptation induced in a manner independent of H(2)O(2)-degrading activity.     

Loss of bone minerals and strength in rats with aldosteronism

Congestive heart failure (CHF) is a clinical syndrome with origins rooted in a salt-avid state largely mediated by effector hormones of the circulating renin-angiotensin-aldosterone system. Other participating neurohormones include catecholamines, endothelin-1, and arginine vasopressin. CHF is accompanied by a systemic illness of uncertain causality. Features include the appearance of oxidative/nitrosative stress and a wasting of tissues including bone. Herein we hypothesized that inappropriate (relative to dietary Na+) elevations in plasma aldosterone (Aldo) contribute to an altered redox state, augmented excretion of divalent cations, and in turn, a loss of bone minerals and strength. In uninephrectomized rats that received chronic Aldo and 1% NaCl treatment for 4-6 wk, we monitored plasma alpha1-antiproteinase activity, which is an inverse correlate of oxidative/nitrosative stress; plasma concentrations of ionized Mg2+ and Ca2+; urinary Mg2+ and Ca2+ excretion; and bone mineral composition and strength to flexure stress. Compared with controls, we found reductions in plasma alpha1-antiproteinase activity and ionized Mg2+ and Ca2+ together with persistently elevated urinary Mg2+ and Ca2+ excretion, a progressive loss of bone mineral density and content with reduced Mg2+ and Ca2+ concentrations, and a reduction in cortical bone strength. Thus the hypermagnesuria and hypercalciuria that accompany chronic Aldo-1% NaCl treatment contribute to the systemic appearance of oxidative/nitrosative stress and a wasting of bone minerals and strength.     

Parathyroid hormone stimulation of the renal 25-hydroxyvitamin D-1alpha-hydroxylase--effect of age and free radicals

The capacity of parathyroid hormone (PTH) to increase serum 1,25(OH)(2)D levels declines with age in both rats and humans. In young rats, PTH stimulates renal 1,25(OH)(2)D production and increases mRNA levels for the terminal mitochondrial P450 of the 1alpha-hydroxylase complex (CYP27B1 or CYP1alpha). However, in older rats PTH increases mRNA levels but not 1,25(OH)(2)D production. This suggests that in old animals there is either decreased CYP1alpha protein levels in response to PTH or that the protein produced lacks functionality. The CYP1alpha protein is located on the inner mitochondrial membrane, the site of increased free radical production with age. To study these possibilities, we examined the effect of PTH and free radicals on CYP1alpha expression in a model system-AOK-B50 renal tubular cells. PTH increased CYP1alpha mRNA and protein in a similar time-dependent manner, suggesting that CYP1alpha protein levels were largely regulated by mRNA levels. The effect of free radicals was determined by preincubation with hydrogen peroxide (H(2)O(2)), a standard model for studying free radical damage. H(2)O(2) inhibited PTH-stimulated CYP1alpha protein levels and 1,25(OH)(2)D production in a dose dependent manner. However, 1,25(OH)(2)D production was more sensitive to H(2)O(2) than was CYP1alpha protein levels. This suggests that the catalytic activity of the CYP1alpha protein may be reduced by free radical damage in these cells. Future studies will focus on detecting oxidative damage in this model system and in vivo.     

Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.     

Evaluation of the ability of the angiotensin-converting enzyme inhibitor captopril to scavenge reactive oxygen species.

Captopril, an inhibitor of angiotensin-converting enzyme, has been suggested to have additional cardioprotective action because of its ability to act as an antioxidant. The rates of reaction of captopril with several biologically-relevant reactive oxygen species were determined. Captopril reacts slowly, if at all, with superoxide (rate constant less than 10(3) M-1 s-1) or hydrogen peroxide (rate constant less than M-1 s-1). It does not inhibit peroxidation of lipids stimulated by iron ions and ascorbate or by the myoglobin/H2O2 system. Indeed, mixtures of ferric ion and captopril can stimulate lipid peroxidation. Captopril reacts rapidly with hydroxyl radical (rate constant greater than 10(9) M-1 s-1) but might be unlikely to compete with most biological molecules for OH because of the low concentration of captopril that can be achieved in vivo during therapeutic use. Captopril did not significantly inhibit iron ion-dependent generation of hydroxyl radicals from hydrogen peroxide. By contrast, captopril is a powerful scavenger of hypochlorous acid: it was able to protect alpha 1-antiproteinase (alpha 1 AP) against inactivation by this species and to prevent formation of chloramines from taurine. We suggest that the antioxidant action of captopril in vivo is likely to be limited, and may be restricted to protection against damage by hypochlorous acid derived from the action of neutrophil myeloperoxidase.     

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.     

The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method.

Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range.     

Purification and characterization of canine alpha-1-antiproteinase.

The principal canine plasma protease inhibitor, alpha-1-antiproteinase, has been purified 90-fold with a 25% yield to apparent homogeneity. The purification scheme includes anion-exchange chromatography, to separate away the bulk of the serum albumin; affinity chromatography by insolubilized concanavalin A, to remove most of the other serum proteins as well as traces of albumin; and, finally, sizing on Sephacryl-S-200. Unique to this purification scheme is the batch use of insolubilized hemoglobin--Sepharose beads to remove the ubiquitous contaminant haptoglobin. The purified material has an apparent molecular weight of 58 000, 11.2% carbohydrate, and an E280nm1% = 5.82, and can be separated by isoelectric focusing into at least two distinct forms with pI values of 4.40 and 4.52. In addition, canine alpha-1-antiproteinase is immunologically distinct from human alpha-1-antiproteinase.     

衰老对AhR 及其血管生成相关因子表达的影响

目的:探讨芳香烃受体(aryl hydrocarbon receptor,Ah R)在过氧化氢(H_2O_2)诱导的人脐静脉内皮细胞(HUVECs)衰老模型中表达的变化情况及其对血管生成相关因子表达的影响。方法:采用不同浓度的H_2O_2(100μM,200μM,300μM)处理HUVECs诱导内皮细胞衰老,通过β-半乳糖苷酶染色观察细胞衰老情况,Western blot检测HUVECs中Ah R蛋白以及血管生成相关因子低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)蛋白表达,ELISA检测细胞培养上清液中血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的变化。结果:与对照组相比,经过不同浓度H_2O_2处理后,HUVECs均出现细胞衰老表现,Ah R蛋白表达显著增加,HIF-1α以及VEGF蛋白表达明显减少,且均呈现出剂量依赖性,以300μM H_2O_2处理组最为显著。结论:在过氧化氢诱导衰老的人脐静脉内皮细胞中Ah R蛋白表达明显增加,HIF-1α和VEGF等促血管生成因子明显减少。     

Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells

Oxidative inactivation of protein tyrosine phosphatases and calcineurin is a well established mechanism; however, little information with regard to the effect of oxidants on PP1 and PP2A activity is available. Herein, we show that PP1 activity is inhibited by H(2)O(2) treatment in differentiated PC12 cells both in vitro and in vivo experiments. Thiol-antioxidant N-acetyl-cysteine (NAC) and reduced glutathione (GSH), when added in vitro to lysates from H(2)O(2)-treated cells, reversed PP1 inhibition. H(2)O(2) treatment increased eIF2 alpha phosphorylated levels (eIF2 alpha P) in a time- and dose-dependent fashion and promoted protein synthesis inhibition. Interestingly, NAC pretreatment protected cells from H(2)O(2)-induced PP1 inactivation and, consequently, it abolished increased H(2)O(2)-induced eIF2 alpha phosphorylation and protein synthesis inhibition. In addition, PP1 inhibitor tautomycin prevented both NAC-induced PP1 reactivation and eIF2 alpha P dephosphorylation in H(2)O(2)-treated cells. Taken together, our findings support a role for PP1 in eIF2 alpha phosphorylation and oxidative stress-triggered translation down regulation.     

Recovery of porcine aortic endothelial cell prostaglandin synthesis following inhibition by sublethal concentrations of hydrogen peroxide

Confluent monolayers of porcine aortic endothelial cells exposed for 10 min to 100 microM H2O2 lose their capacity to produce prostaglandins in response to addition of saturating exogenous arachidonic acid. Significant recovery of prostaglandin I2 and E2 synthesis occurred within 3 h and full enzymatic capacity returned by 6 h. Reducing the injury by exposure to half the amount of H2O2 allowed prostaglandin I2 production to recover to a greater extent in 3 h, while cells exposed for 60 min to either 0.5 or 1.0 mM H2O2 demonstrated no recovery. Pre-treatment with either actinomycin D or cycloheximide also prevented recovery following exposure to 100 microM peroxide. Injured cells did not recover when incubated with balanced salts after removal of peroxide, while incubation with medium 199 allowed for the complete return of synthetic capacity. Addition of 1% fetal calf serum in medium 199 did not facilitate recovery. Production of prostaglandins from endogenous arachidonic acid, released by either bradykinin or the ionophore A23187, was also inhibited by H2O2 exposure, however, full recovery of this stimulated synthesis occurred within 3 h. Cycloheximide pre-treatment completely inhibited recovery of bradykinin-induced prostaglandin I2 synthesis. These data demonstrate that sublethal concentrations of H2O2 irreversibly inactivate fatty acid cyclooxygenase and that synthesis of new enzyme is required for recovery. This return of activity occurs more rapidly for production of prostaglandins from endogenous arachidonic acid compared with production following addition of exogenous substrate.     

Inhibition of hypochlorous acid-induced oxidative reactions by nitrite: is nitrite an antioxidant?

Acute and chronic inflammation result in increased nitrogen monoxide (z.rad;NO) formation and the accumulation of nitrite (NO(2)(-)). Neutrophils stimulated by various inflammatory mediators release myeloperoxidase to produce the cytotoxic agent hypochlorous acid (HOCl). At physiologically attainable concentrations, we found that NO(2)(-) significantly inhibits HOCl-mediated DNA strand breakage and ascorbate depletion. HOCl-mediated inactivation of pure alpha(1)-antiproteinase or of the elastase inhibitory capacity of human plasma was inhibited by the addition of NO(2)(-). NO(2)(-) was more effective than ascorbate, GSH, and urate at inhibiting HOCl-mediated toxicity to human HepG2 cells in culture. These data suggest that NO(2)(-) may act in an antioxidant manner by removing HOCl at sites of inflammation where both HOCl and z.rad;NO are overproduced.     

Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin.

Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein. The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta. Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+). Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect. Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time. Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta. In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups. Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+. Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M. Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE. Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta. Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M. The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent. These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS)