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PubMed1999

Evaluation of the effects of Gd complexes used as magnetic resonance imaging contrast agents, on superoxide dismutase: comparison of two methods.

Investigations are currently being made into the safety of gadolinium complex contrast agents used in Magnetic Resonance Imaging. Their hyperosmolality or potential Gd3+ release is evoked as a cause of various anaphylactoid reactions to be observed in humans after intravenous injection. An estimation has already been made of their effects on the liberation of reactive oxygen intermediates by neutrophils. The purpose of this study was to find a suitable method to measure SOD activity in the presence of these hyperosmolar solutions, and to evaluate their action on this activity. Two techniques were compared to measure this activity. Results and statistical analysis showed that pyrogallol autoxidation was greatly affected by solution osmolalities, whereas ferricytochrome C reduction was not. Gadopentetate dimeglumine and gadoterate meglumine seemed to activate Cu, Zn SOD in vitro, but did not exhibit any SOD-like activity. Gadodiamide did not interfere with this system of detoxication.

Contrast Media↗
PubMed1997

Cobalt chloride and desferrioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species.

We have recently proposed a H2O2-generating b-type cytochrome as part of the cellular oxygen sensor that controls O2-dependent erythropoietin (Epo) production in the human hepatocellular carcinoma cell line HepG2. H2O2 could act as an intracellular signaling molecule because its production in HepG2 cells is strictly dependent on the pericellular PO2. High cellular levels of H2O2 inhibit hypoxia-induced Epo production while low levels-as under hypoxic conditions-allow full expression of the Epo gene. Since cobalt chloride (CoCl2) and the iron chelator desferrioxamine (DSF) both mimic the hypoxic induction of Epo production we studied the influence of CoCl2 and DSF on the formation and on the action of reactive O2-species with respect to Epo production. Both chemicals reduced the H2O2-dependent 123-dihydrorhodamine fluorescence in HepG2 cells. The inhibition of Epo production by exogenous H2O2 was completely antagonized by DSF. This might indicate that H2O2 exerts its inhibition through a Fenton type reaction. On the other hand, NADPH and pyrogallol which stimulate the production of O2- inhibited Epo production. CoCl2 antagonized their effects. From our results we propose different sites of interaction with the putative signaling chain for DSF and CoCl2. While DSF appears to reduce the action of the H2O2 molecule, CoCl2 might act further upstream through the induction of H2O2-scavenger systems or by interfering with its production.

Cell Line↗
PubMed1998

S-nitrosothiols and the nitrergic neurotransmitter in the rat gastric fundus: effect of antioxidants and metal chelation.

1. The effects of the antioxidants ascorbic acid and alpha-tocopherol and of the metal chelator ethylenediaminetetraacetic acid (EDTA) were studied on relaxations in response to S-nitrosothiols, authentic nitric oxide (NO) and nitrergic non-adrenergic non-cholinergic stimulation of the rat gastric fundus. 2. The S-nitrosothiols S-nitrosocysteine (1-100 nM), S-nitrosoglutathione (0.01-3 microM) and S-nitroso-N-acetylpenicillamine (0.01-3 microM) induced concentration-dependent relaxations of the rat gastric fundus muscle strips, which were precontracted with prostaglandin F2alpha. The relaxations to all S-nitrosothiols were concentration-dependently enhanced by the antioxidants ascorbic acid (0.1-3 microM) and alpha-tocopherol (3-30 microM) and inhibited by the metal chelator EDTA (26 microM). 3. Ascorbic acid and alpha-tocopherol alone did not induce a relaxation of the precontracted rat gastric fundus muscle strip. However, when ascorbic acid (1 microM) or alpha-tocopherol (1 microM) were injected in the organ bath 1 minute after S-nitrosoglutathione (0.1 microM) or after S-nitroso-N-acetylpenicillamine (0.1 microM), they induced an immediate, sharp and transient relaxation. This relaxation was inhibited by the superoxide generator pyrogallol (2 microM). Such a relaxation to ascorbic acid or alpha-tocopherol was not observed in the presence of S-nitrosocysteine (10 nM). 4. Electrical field stimulation (0.5-4 Hz) of the precontracted rat gastric fundus strips induced frequency-dependent nitrergic relaxations which were mimicked by authentic NO (3-300 nM) and by acidified sodium nitrite NaNO2 (0.3-10 microM). Ascorbic acid (0.33-3 microM), alpha-tocopherol (3-30 microM) or EDTA (26 microM) did not affect the relaxations to nitrergic stimulation, NO or NaNO2. 5. In summary, relaxations to S-nitrosothiols in the rat gastric fundus are enhanced by the antioxidants ascorbic acid and alpha-tocopherol and inhibited by the metal chelator EDTA. However, relaxations to nitrergic stimulation of the rat gastric fundus or those to authentic NO were not affected by the antioxidants or by the metal chelator. These results indicate that antioxidants and metal chelators have a different effect on the biological activity of S-nitrosothiols and on that of the nitrergic neurotransmitter. Therefore, our results suggest that S-nitrosothiols do not act as intermediate compounds in nitrergic neurotransmission in the rat gastric fundus.

Animals↗
PubMed1999

Tyrosine nitration in blood vessels occurs with increasing nitric oxide concentration.

1. Experiments were designed to explore the effects of nitric oxide (NO) donors on generation of superoxide (O2.-) and peroxynitrite (ONOO-) in rabbit aortic rings. 2. Following inhibition of endogenous superoxide dismutase (SOD), significant basal release of O2.- was revealed (0.9 +/- 0.01 x 10(-12) mol min-1 mg-1 tissue). Generation of O2.- increased in a concentration-dependent manner in response to NADH or NADPH (EC50 = 2.34 +/- 1.18 x 10(-4) and 6.21 +/- 1.79 x 10(-3) M respectively, n = 4). NADH-stimulated O2.- chemiluminescence was reduced by approximately 85% in the presence of exogenous SOD (15 x 10(3) U ml-1). 3. Incubation of aortic rings with S-nitrosoglutathione (GSNO; 1 x 10(-5)-3 x 10(-3) M) or sodium nitroprusside (SNP; 1 x 10(-8)-1 x 10(-3) M), resulted in a concentration-dependent quenching of O2.- chemiluminescence which was proportional to NO release. 4. ONOO- formation was assessed indirectly by determining protein tyrosine nitration in rabbit aorta using a specific antibody against nitrotyrosine. Basally and in the presence of NADH, a single band was detected. Incubation of aortic rings with either GSNO (1 x 10(-3) M) alone or GSNO with NADH resulted in the appearance of additional nitrotyrosine bands. Incubation of serum albumin with GSNO alone did not cause nitrotyrosine formation. In contrast, incubation with 3-morpholinosydonomine (SIN-1; 1 x 10(-3) M, 10 min), resulted in marked nitration of albumin which was reduced by oxyhaemoglobin or SOD. Incubation of albumin with GSNO and pyrogallol, a O2.- generator, also resulted in protein nitration. 5. Addition of exogenous NO results in nitrotyrosine formation in rabbit aortic rings. Nitrotyrosine formation is likely to result from the reaction of exogenous NO and basal endogenous O2.- resulting in the formation of ONOO-. Formation of ONOO- and nitration of tyrosine residues potentially could lead to vascular damage and might represent unexpected adverse effects of long-term nitrate therapy.

Animals↗
PubMed2000

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra.

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoine-1-oxyl 3-oxide (carboxy-PTIO), the specific guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 microM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml(-1)) inhibited NO-mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy-PTIO (0.1 - 0.5 mM) inhibited NO-mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml(-1)). Relaxations to NO were reduced, but not abolished, by ODQ (10 microM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB, 0. 5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N-ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L-cysteine (L-cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD-independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open. British Journal of Pharmacology (2000) 129, 53 - 62

Animals↗
PubMed1998

Effect of Korean red ginseng on the rabbit corpus cavernosal smooth muscle.

The extracts of Korean red ginseng (EKG) is a complex mixture containing ginsenosides, polysaccharides, and several other products. Animal experiments have shown that the intravenous administration of extract of ginseng reduces blood pressure. Recently, it has been reported that ginseng has a relaxing effect on vascular smooth muscle and that the relaxation is associated with nitric oxide (NO) released from the vascular endothelium. The present study was undertaken to investigate the effects of EKG on isolated rabbit corpus cavernosal smooth muscle for evaluation of the possibility of developing EKG as an pharmacoerecting agent. Strips of rabbit corpus cavernosum were mounted in organ chambers to measure isometric tension. On the precontracted muscle strip with phenylephrine (PHE; 5 x 10(-6) M), EKG began to exert a relaxing effect at the concentration of 1 mg/ml and the maximal relaxation effect was reached at 40 mg/ml in a dose-dependent manner. EKG was inhibited significantly by endothelial disruption and by pretreatment with methylene blue, pyrogallol, L-NNA or atropine. EKG partially inhibited the PHE (5 x 10(-6) M) induced contraction up to 45.67% of the control in a dose-dependent fashion. EKG decreased basal tension as well as inhibited the contraction induced by addition of CaCl2 (10(-3) M) dose-dependently in muscle strips at basal equilibrated state in Ca2+ free, high K+ depolarizing solution. EKG also inhibited the contraction induced by depolarization with 20, 40 and 60 mM of KCl. However, this inhibitory effect did not occur with high concentrations of KCl (80 and 120 mM). EKG has a relaxing effect on the rabbit corpus cavernosal tissue in a dose dependent manner. The relaxation action of EKG is mediated by multiple action mechanisms that include increasing the release of NO from the corporal sinusoids, increasing intracellular calcium sequestration, and a hyperpolarizing action.

Animals↗
PubMed2003

A mesh microcontactor for 2-phase reactions.

The design, fabrication and use of mesh microcontactor structures is described. These allow contact between immiscible fluid phases (liquid/liquid or gas/liquid) enabling mass transfer and reaction between and within the phases. The phases are not mixed and are removed separately for subsequent use or analysis. The structures were designed for kinetic studies on biphasic reactions with the ability to handle sequential samples without excessive sample dispersion. Mass transport conditions are defined by fluid layer depths of 100 microm and the volume for each phase contacting in the reaction region was selected at 100 microl as sufficient for a range of analytical techniques. Micromeshes with pore diameter, depth, and spacing each of approximately 5 microm were formed by electrodeposition of nickel onto substrates with defined photoresist layers, and released by etching a copper sub-layer. Reactor enclosures defining chambers on each side of the mesh were formed from milled glass and metal components. The assembled structures have been used in flow-through and static fluid modes. System function has been demonstrated for both liquid/liquid and gas/liquid reactions. Test chemistries selected for ease of optical monitoring were hydrolysis of colourless fluorescein diacetate in toluene with transfer as fluorescein anion to aqueous alkali, and oxygen absorption into aqueous alkaline pyrogallol solution generating a coloured product, purpurogallin.

Journal Article↗
PubMed2001

The role of distal tryptophan in the bifunctional activity of catalase-peroxidases.

Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).

Bacterial Proteins↗
PubMed2000

Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum.

Tyrosinase, with an isoelectric point at pH 4.9, was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. Gel filtration, N-terminal amino acid sequencing and SDS/PAGE analysis indicate that T. roseum tyrosinase is composed of two identical subunits, each with a molecular mass of 43000 Da. The enzyme exhibited high substrate specificity towards catechol, chlorogenic acid, L-3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) and pyrogallol. The K(m) value of the enzyme for L-DOPA was 0.18 mM. beta-Mercaptoethanol and sodium diethyldithiocarbamate notably inhibited the enzymic activity. The activity of the enzyme was optimal at pH 9.5 and 70 degrees C, and was increased by addition of 1 mM Mg(2+), K(+) or Cu(2+). The enzyme was highly stable against high temperature and guanidine hydrochloride. The N-terminal amino acid sequence of the enzyme was determined to be Asp-Ile-Asn-Gly-Gly-Gly-Ala-Thr-Leu-Pro-Gln-Lys-Leu-Tyr. These facts indicate that T. roseum tyrosinase appears to be distinct from the tyrosinases so far purified from other sources.

Amino Acid Sequence↗
PubMed1966

The purification and some properties of the polyphenol oxidase from tea (Camellia sinensis L.).

1. Polyphenol oxidase (EC 1. 10. 3.-) from the shoots of the tea plant was purified about 5000-fold on a dry-weight basis. 2. At an intermediate stage of purification four soluble yellow fractions were obtained. They are believed to represent complexes of a basic enzyme protein with acidic phenolic oxidation products and nucleic acids. After removal of the complex-forming materials the fractions were blue and similar to each other. About 40% of the activity could not be extracted from the acetone-dried powder. 3. Each of the four blue fractions was resolved further into two species, A and B. The following results refer to species A. 4. The enzyme showed absorption maxima at 279mmu (E(1%) (1cm.), 13.5) and 611mmu (E(1%) (1cm.), 0.84) with a shoulder at 330mmu. The enzyme was bleached by substrate under anaerobic conditions and the colour was restored by oxygen. 5. The molecular weight measured by sedimentation and diffusion was 144000+/-16000. The copper content was 0.32% (w/w). 6. Kinetic constants are given for a number of substrates and inhibitors, including the natural substrates of the tea leaf. The specific activity towards pyrogallol was 373 units/mg. at 30 degrees . 7. The best substrates were o-dihydric phenols. Quinol and p-phenylenediamine were slowly oxidized. Monohydric phenols and ascorbic acid were not oxidized. 8. The kinetics of oxidation of most substrates are consistent with a mechanism in which oxidized and reduced forms of the enzyme form binary complexes with phenol and oxygen respectively. A modified mechanism is postulated for the oxidation of chlorogenic acid. 9. The relation of the results to the mechanism of tea fermentation is discussed.

Journal Article↗
PubMed1966

A rapid assay for peroxidase activity.

1. Peroxidase has been assayed by a chronometric method involving the coupled reaction of ascorbic acid with the product of the enzymic action on benzidine. 2. Measurements of the activities of horseradish and tea peroxidase by this and two other methods, involving respectively pyrogallol and o-dianisidine, are compared. 3. It is claimed that the chronometric method is relatively simple, rapid and accurate. 4. The method can be used in the presence of polyphenol oxidases.

Journal Article↗
PubMed1980

Bromoperoxidases from Penicillus capitatus, Penicillus lamourouxii and Rhipocephalus phoenix.

The isolation and purification of bromoperoxidases from three marine subtropical green algae is described. In the presence of KBr and H2O2, each halide-specific enzyme catalyses the bromination of monochlorodimedone (2-chloro-5,5-dimethylcyclohexane-1,3-dione) to bromochlorodimedone (2-bromo-2-chloro-5,5-dimethylcyclohexane-1,3-dione). The enzymes also catalyse the oxidation of pyrogallol, o-phenylenediamine and I- to I3-. Preliminary characterization of these enzymes reveals acidic pH optima, high thermal stability, sensitivity to higher H2O2 concentrations, and apparent molecular weights ranging from 48000 to 60000.

Bromides↗
PubMed1980

Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi.

The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals.

Animals↗
PubMed1991

Characterization and immunological properties of selenium-containing glutathione peroxidase induced by selenite in Chlamydomonas reinhardtii.

The selenite-induced glutathione peroxidase in Chlamydomonas reinhardtii has been purified about 323-fold with a 10% yield, as judged by PAGE. The native enzyme had an Mr of 67,000 and was composed of four identical subunits of Mr 17,000. Glutathione was the only electron donor, giving a specific activity of 193.6 mumol/min per mg of protein. L-Ascorbate, NADH, NADPH, pyrogallol, guaiacol and o-dianisidine did not donate electrons to the enzyme. In addition to H2O2, organic hydroperoxides were reduced by the enzyme. The Km values for glutathione and H2O2 were 3.7 mM and 0.24 mM respectively. The enzyme reaction proceeded by a Ping Pong Bi Bi mechanism. Cyanide and azide had no effect on the activity. The enzyme contained approx. 3.5 atoms of selenium per mol of protein. On immunoprecipitation, Chlamydomonas glutathione peroxidase was precipitated and its activity was inhibited about 90% by the antibody raised against bovine erythrocyte glutathione peroxidase. The antibody also cross-reacted with the subunits of Chlamydomonas glutathione peroxidase in Western blotting SDS/PAGE. In terms of enzymic, physico-chemical and immunological properties, the experimental results demonstrate clearly that Chlamydomonas glutathione peroxidase resembles other well-characterized glutathione peroxidases from animal sources that contain selenium.

Animals↗
PubMed1993

Purification and partial characterization of lipoxygenase with dual catalytic activities from human term placenta.

Lipoxygenase possessing dual catalytic activities, i.e. dioxygenase and hydroperoxidase, was purified from the cytosols of term placentas from non-smoking women. Concanavalin A affinity chromatography followed by phenyl-Sepharose CL-4B chromatography resulted in the separation of one hydrophobic and one non-hydrophobic isoenzyme. The concanavalin A-purified enzyme was used in all subsequent experiments. The dioxygenase activity of the enzyme exhibited a Vmax. of 204.37 +/- 17.66 nmol/min per mg of protein and a Km of 0.79 mM for linoleic acid. The involvement of dioxygen in enzymic linoleic acid oxidation was confirmed by O2 uptake studies. Arachidonic acid and linolenic acid also served as substrates for the dioxygenase activity. The placental lipoxygenase co-oxidized benzidine in the presence of linoleic acid (hydroperoxidase activity). Both the dioxygenase and hydroperoxidase activities were significantly stimulated by Ca2+ (1-100 microM), ATP (10-400 nM) and H2O2 (1-10 nM). Similarly, these two activities were inhibited by nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, gossypol, esculetin, butylated hydroxyanisole and butylated hydroxytoluene. Boiled enzyme was without significant dioxygenase and hydroperoxidase activities. Pyrogallol, 3,3'-dimethoxybenzidine, 3,3',5,5'-tetramethylbenzidine, tetramethylphenylenediamine and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) were also co-oxidized by the placental lipoxygenase. These results suggest that: (i) lipoxygenase from human term placenta exhibits both dioxygenase and hydroperoxidase activities, and (ii) this enzyme represents an important pathway for chemical oxidation in the placentas of non-smoking women.

Catalysis↗
PubMed1994

Effects of oxidative stress on expression of extracellular superoxide dismutase, CuZn-superoxide dismutase and Mn-superoxide dismutase in human dermal fibroblasts.

To determine the effect of oxidative stress on expression of extracellular superoxide dismutase (EC-SOD), CuZn-SOD and Mn-SOD, two fibroblast lines were exposed for periods of up to 4 days to a wide concentration range of oxidizing agents: xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, alpha-naphthoflavone, hydroquinone, catechol, Fe2+ ions, Cu2+ ions, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure. The cell lines were cultured both under serum starvation and at a serum concentration that permitted growth. Under no condition was there any evidence of EC-SOD induction. Instead, the agents uniformly, dose-dependently and continuously reduced EC-SOD expression. We interpret the effect to be due to toxicity. Enhancement of the protection against oxidative stress by addition of CuZn-SOD, catalase and low concentrations of selenite did not influence the expression of any of the SOD isoenzymes. Removal of EC-SOD from cell surfaces by heparin also did not influence SOD expression. Mn-SOD was moderately induced by high doses of the first 11 oxidants. Apart from reduction at high toxic doses, there were no significant effects on the CuZn-SOD activity by any of the treatments. Thus EC-SOD, previously shown to be profoundly influenced by inflammatory cytokines, was not induced by its substrate or other oxidants. In a similar fashion, Mn-SOD, previously shown to be greatly induced and depressed by cytokines, was only moderately influenced by oxidants. We suggest that the regulation of these SOD isoenzymes in mammalian tissues primarily occurs in a manner co-ordinated by cytokines, rather than as a response of individual cells to oxidants.

Cell Line↗
PubMed1996

The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli.

Synechococcus PCC 7942, a cyanobacterium, possesses catalaseperoxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2 +/- 0.27 mM and the kcat value was 2.6 x 10(4) s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3 +/- 0.84 and 20.2 +/- 0.95 microM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase-peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase-peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase-peroxidases of prokaryotic cells. Escherichia coli BL21 (DE3)plysS, harbouring a recombinant plasmid containing the catalase-peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

Amino Acid Sequence↗
PubMed1997

Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles.

Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.

Binding Sites↗