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PubMed1979

Mammalian endogenous peroxidases as cellular markers and as biosynthetic endpoints of hormone-mediated activity: viewpoint from cytochemistry.

Aldehyde-resistant, diaminobenzidine-stained endogenous peroxidases form ideal markers for the biochemical endpoints of hormone stimulation and differentiation of certain mammalian cells and tissues. The lactoperoxidase (LPO)-type of endogenous peroxidases are synthesized by the acinar cells of the salivary, Harderian, lacrimal and mammary glands and are present in their secretions. These LPO-type enzymes, that are inhibited by cyanide and aminotriazole, appear to operate extracellularly as bactericidal agents in milk and in other biological fluids. In the mammary gland, lactoperoxidase is a consistent marker enzyme for differentiated acinar cells engaged in lactogenesis. Myeloperoxidase (MPO)-type endogenous peroxidases are prominent markers for the GERL endomembrane system and differentiated lysosomes in certain cells of the reticuloendothelial system and phagocytes. MPO is prominent within eosinophils, peritoneal macrophages and in Kupffer cells. The MPO-type endogenous peroxidases function primarily within lysosomes as bactericidal agents. Thyroid peroxidase (TPO) is relegated to the cisternae of the granular endoplasmic reticulum and Golgi apparatus, to apical cytoplasmic vesicles and to the luminar cell membrane surface of acinar cells. The enzyme is probably activated at release and functions both in the organification reaction (T leads to To) and in the biosynthesis of thyroxine. Thyroid stimulating hormone (TSH) appears to play a key role in the regulation of TPO levels and activity in the thyroid gland. Certain tissues displaying growth-dependency on estrogen (i.e., uterus, cervix, vagina and the DMBA-induced rat mammary tumor) synthesize and secrete endogenous peroxidase into their lumina. These enzymes serve as important marker proteins of estrogen action, in that they occur distal to the binding of estrogen to its receptor protein. Estrogen antagonists, particularly CI-628 (Parke-Davis) and Nafoxidine (Upjohn) that appear to function through the estrogen receptor mechanism, also induce synthesis of the reproductive tract endogenous peroxidase but inhibit growth of these tissues. Progesterone antagonizes the synthesis of the reproductive tract peroxidases and inhibits growth of the tissues as well, in part, through the reduction of the cytosol estrogen receptor protein. Endogenous peroxidase activity appears to represent a reliable marker for rodent breast cancer tissues displaying dependency for estrogen and is of potential interest as a diagnostic marker protein in human breast cancer. Rat uterine peroxidase (UP) has been investigated by microelectrophoretic techniques. The molecular weight of UP has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergents. The isoelectric point of UP is located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, UP was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000.

Animals↗
PubMed1993

Influence of cellobiose oxidase on peroxidases from Phanerochaete chrysosporium.

Reduction of H2O2-oxidized manganese peroxidase (MnP), lignin peroxidase and, to some extent, horseradish peroxidase, was studied in the presence of cellobiose oxidase (CbO) and cellobiose. It was found that the reversion rates for MnP compound II and lignin peroxidase compound II back to native enzymes increased significantly in the presence of CbO and cellobiose. However, the reduction of cytochrome c by CbO plus cellobiose was 40 times faster than the reduction of MnP compound II. Also, the lag phase before reversion to the native states decreased for all three peroxidases in the presence of CbO and cellobiose. Active CbO did not repress formation of compounds I or II of the peroxidases, and Mn2+/veratryl alcohol reduced compound II of the peroxidases much more rapidly than did active CbO. This indicates that, in the presence of Mn2+ or veratryl alcohol, MnP and lignin peroxidase can complete their catalytic cycles and function normally without interference from CbO. Without the presence of peroxidase substrates, active CbO reduced compound II of the above peroxidases.

Basidiomycota↗
PubMed1986

Direct measurements of intramolecular electron transfer rates between cytochrome c and cytochrome c peroxidase: effects of exothermicity and primary sequence on rate.

Rapid mixing of ferrocytochrome c peroxidase [cyt c peroxidase(II)] and ferricytochrome c [cyt c(III)] results in the reduction of cyt c(III) by cyt c peroxidase(II). In 10 mM phosphate, pH 7.0, the rate of decay of cyt c peroxidase(II) and the rate of accumulation of cyt c(II) give equal first-order rate constants: k = 0.23 +/- 0.02 s-1. Equivalent results are obtained by pulse radiolysis using isopropanol radical as the reducing agent. This rate is independent of the initial cyt c(III):cyt c peroxidase(II) ratios. These results are consistent with unimolecular electron transfer occurring within a cyt c(III)-cyt c peroxidase(II) complex. When cyt c is replaced by porphyrin cyt c (iron-free cyt c), a complex still forms with cyt c peroxidase. On radiolysis, using e-aq as the reducing agent, intracomplex electron transfer occurs from the porphyrin cyt c anion radical to cyt c peroxidase(III) with k = 150 s-1. This large rate increase with increasing delta G degrees suggests that the barrier for intracomplex electron transfer is large. Finally, we have briefly investigated how the cyt c peroxidase(II)----cyt c(III) rate depends on the primary structure of cyt c(III). We find the reactivity order to be as follows: yeast (k = 3.4 s-1) greater than horse (k = 0.3 s-1) greater than tuna (k = 0.2 s-1). These results mirror a report [Ho, P. S., Sutoris, C., Liang, N., Margoliash, E. & Hoffman, B. M. (1985) J. Am. Chem. Soc. 107, 1070-1071] on excited state reactions of the cyt c/cyt c peroxidase couple.

Amino Acid Sequence↗
PubMed1985

Superoxide dismutase and peroxidase are coordinately regulated in differentiated and transformed tissues of Nicotiana tabacum.

We used a series of normal and Agrobacterium-transformed, bacteria-free tobacco tissue cultures which differ in their levels of histodifferentiation to test the relationship of superoxide dismutase, peroxidase, and catalase to oncogenic transformation and differentiation. When compared with normal callus, tumor callus contained reduced levels of both superoxide dismutase and peroxidase, and a reduced number of isozymes of both enzymes. Teratomas characterized by limited but abnormal differentiation showed increases in superoxide-dismutase activity and isozymes, but levels of peroxidase activity lower than those found in normal callus despite an increase in the number of peroxidase isozymes. A similar disparity between low peroxidase activity and high isozyme number in the shoot suggests that there are increased levels of peroxidase inhibitors or of molecules which interfere with the spectrophotometric assay for peroxidase in more differentiated tissues. As judged by the number of isozymes of both superoxide dismutase and peroxidase in each tissue, the following conclusions are warranted: first, tobacco copper/zinc superoxide dismutases and peroxidases are encoded in several duplicated loci which are regulated independently. Second, transformation is associated with a decrease in both the specific activity and isozyme number of superoxide dismutase. Third, the partial release from the total inhibition of expression of differentiated function exhibited by teratoma is associated with an increase in both the activity and isozyme number of superoxide dismutase. Finally, the expression of superoxide dismutase and peroxidase isozymes appears to be coordinated during differentiation in a manner that is consistent with their role in an integrated mechanism for the removal of reduced oxygen species.

Catalase↗
PubMed1981

Substituted hemins as probes for structure-function relationships in horseradish peroxidase.

Low temperature visible spectra of Compounds I from peroxidases reconstituted with protohemin, 2-formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and 2,4-diacetyldeuterohemin reveal that these Fe(IV) porphyrin pi-cation radicals take the 2A2u of peroxidase-type electronic ground state. Compound I of deuterohemin horseradish peroxidase, however, takes the 2A1u or catalase type pi-cation radical electronic ground state. Since deuterohemin horseradish peroxidase possesses no catalase activity, the structure of the peroxidase apoprotein (other than those factors which might influence the Compound I pi-cation radical ground state) is concluded to play the major role in determining the reactivity of Compound I toward hydrogen donors. Studies on peroxidases substituted with the hemins 2-formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and mesohemin reveal that isoelectronic hemins differentially interact with the peroxidase apoprotein. The hemin 2- and 4-substituents are therefore concluded to interact sterically with the horseradish peroxidase apoprotein. While a variety of 2- and 4-substituted hemins were observed to bind rapidly with apo horseradish peroxidase to form active substituted enzymes, small changes in the substituents in the 6- and 7-positions had drastic effects on the rates of binding to apoperoxidase and the activities of the reconstituted enzymes. Even addition of a single methylene to form butyrate instead of propionate side chains drastically altered the rate of binding of the hemin and the activity of the substituted enzyme. It therefore appears that while the 2-, 4-, 6-, and 7-substituents of the hemins in horseradish peroxidase all interact with the protein, the polypeptide chain possesses more conformational flexibility in the area which binds the 2- and 4-substituents.

Heme↗
PubMed1994

Tubulovesicular transport of horseradish peroxidase in isolated rat hepatocyte couplets: effects of low temperature, cytochalasin B and bile acids.

The transcytotic vesicular pathway in isolated rat hepatocyte couplets was investigated using horseradish peroxidase. Ten to 20 min after horseradish peroxidase labeling, vesicles and tubules containing horseradish peroxidase were observed to be predominantly around the bile canaliculi. In hepatocytes incubated in a 4 degrees C medium for 10 min after horseradish peroxidase labeling, few horseradish peroxidase-containing structures were observed around the bile canaliculi, and the fine reticular immunofluorescence of microtubules was reduced. Cells treated with cytochalasin B (a microfilament inhibitor) showed a fair number of horseradish peroxidase-containing structures around the markedly dilated bile canaliculi and the distribution of microtubules was preserved. Cells labeled by horseradish peroxidase and then incubated for 10 min in a horseradish peroxidase-free medium containing 50 mumol/L of taurocholic acid, ursodeoxycholic acid or tauroursodeoxycholic acid had more tubular structures containing horseradish peroxidase around the bile canaliculi than control cells, whereas 50 mumol/L of taurochenodeoxycholic acid, taurodeoxycholic acid, dehydrocholic acid and taurodehydrocholic acid each failed to increase the number of tubular structures. These findings show that horseradish peroxidase was transported in hepatocyte couplets from the cell periphery to the bile canalicular front through the tubulovesicular pathway, depending on cytoplasmic microtubules. Cytoplasmic microfilaments appeared to play a minor role in this transport. Several specific bile acids such as taurocholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid each promoted the tubular transformation.

Animals↗
PubMed1994

Peroxidase activities in bull spermatozoa.

The present study was carried out to determine the localization of peroxidase activity in bull spermatozoa. 3,3'-Diaminobenzidine (DAB) was used as a substrate for revealing peroxidase activity, and light and electron microscopic analysis of the results obtained was performed. Peroxidase activity was detected in the mitochondria of the middle piece and the outer acrosomal membrane. Catalase was excluded as an enzyme, catalyzing the detected peroxidase activity. Concerning the biochemical properties of bull sperm peroxidases, peroxidase activity was found to be manifested in a large pH range, 4-10.5. Bull sperm peroxidase activity appeared to be temperature sensitive and azide sensitive and could be readily inhibited by phenylhydrazine. Electrophoretic analysis of the proteins from bull sperm extracts separated in a Davis-Ornstein system of 7% polyacrylamide gel, followed by the determination of peroxidase activity on the polyacrylamide gels, revealed that all 14 sperm protein fractions available on the gel possessed peroxidase when benzidine was used as a substrate. The possible reasons for the electrophoretic heterogeneity of bull sperm peroxidases are discussed.

3,3'-Diaminobenzidine↗
PubMed1997

Further studies on the inactivation by sodium azide of lignin peroxidase from Phanerochaete chrysosporium.

Azide ion is a mechanism-based inactivator of horseradish peroxidase [Ortiz de Montellano et al. (1988) Biochemistry 27, 5470-5476] and the peroxidase from the coprophilic fungus Coprinus macrorhizus [DePillis and Ortiz de Montellano (1989) Biochemistry 28, 7947-7952]. These peroxidases mediate the one-electron oxidation of azide ion-forming azidyl radical. Inactivation of these enzymes is caused by covalent modification of the heme prosthetic groups by azidyl radical. Lignin peroxidases from the wood-rotting fungus Phanerochaete chrysosporium are also inactivated when they catalyze oxidation of azide ion [Tuisel et al. (1991) Arch. Biochem. Biophys. 288, 456-462; DePillis et al. (1990) Arch. Biochem. Biophys. 280, 217-223]. Following inactivation of horseradish peroxidase and the peroxidase from C. macrorhizus substantial amounts of azidyl-heme adducts have been found. Only trace amounts of such adducts have been found following azide-mediated inactivation of lignin peroxidase. Nevertheless, we have shown that during oxidation of azide by lignin peroxidase H8 destruction of heme occurred and a substantial fraction of the enzyme is irreversibly inactivated. However, the rest of the enzyme forms a relatively stable ferrous-nitric oxide (NO) complex. Although this complex appears to be an inactivated form of the enzyme, we have shown that, when present as the ferrous-NO complex, the enzyme is actually protected from inactivation. The lignin peroxidase ferrous-NO complex reverts slowly (t1/2 = 6.3 x 10(3) s) to the ferric form. Reversion is accelerated if the complex is chromatographed on a PD-10 (Sephadex G-25) column or if veratryl alcohol is added. If azide and hydrogen peroxide (a required cosubstrate) are present (or added), the enzyme undergoes another cycle of catalysis and further inactivation. A detailed reaction mechanism is proposed that is consistent with our experimental observations, the chemistry of azide, and our current understanding of peroxidases.

Azides↗
PubMed1976

The effect of alloxan on the permeability of isolated pancreatic islets to horseradish peroxidase.

The effect of alloxan on the permeability of isolated pancreatic islets to horseradish peroxidase was studied by a perifusion system, which allowed simultaneous monitoring of glucose-induced insulin secretion. Rat islets were perifused with a 5 mg/ml glucose solution containing horseradish peroxidase for ten or thirty minutes, with or without prior exposure to alloxan (20 mg%) for five minutes. Control islets without alloxan treatment showed few necrotic beta cells diffusely infiltrated with exogenous peroxidase; and, the majority were intact beta cells containing numerous peroxidase-positive vesicles. Islets purifused with alloxan in a 1 mg/ml glucose solution showed many damaged beta cells loaded with peroxidase; whereas, intact beta cells contained a few peroxidase-positive vesicles. In islets purifused with alloxan in a 5 mg/ml glucose solution, the features seen in the control islets and the islets purifused with alloxan in a 1 mg/ml glucose solution were observed--namely, degenerated beta cells loaded with peroxidase and intact beta cells with numberous peroxidase-positive vesicles. As the guinea pig is known to be relatively resistant to the effect of alloxan, the effect of alloxan was examined in the isolated islets of guinea pig. The degenerated peroxidase-positive beta cells were not observed in guinea pig islets, which had been exposed to alloxan (20 mg%). It is concluded that the direct alloxan action on rat beta cells is at least partially to damage beta cell membranes with resultant enhanced permeability to horseradish peroxidase.

Alloxan↗
PubMed2000

Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth.

It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 microg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immunoreactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by Mr-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth.

Cell Wall↗
PubMedDate not supplied

The role of selenium peroxidases in the protection against oxidative damage of membranes.

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.

Antioxidants↗
PubMed1992

Ultrastructural localization of peroxidase in atherosclerotic lesions of pigeons.

Atherosclerotic lesions are known to have metabolic alterations which are associated with progressive lipid accumulation. Among the changes, lysosomal enzyme activity has been extensively characterized and at the ultrastructural level has been correlated with the amount of foam cell lipid. In a fashion paralleling lysosomal change, artery wall peroxidase activity is also altered during disease progression. The present study focuses upon the ultrastructural localization of peroxidase activity in atherosclerotic lesions of the aorta and coronary arteries from White Carneau pigeons fed a cholesterol-supplemented (0.3%) diet for 3 years. This resulted in fibrous lesions, rich in smooth muscle cells. The birds were necropsied by perfusion fixation, and peroxidase cytochemistry was carried out using the diaminobenzidine reaction. Peroxidase activity was found within endothelial cells and smooth muscle cells in both the media and intima, but cytochemically demonstrable activity was not found in macrophage foam cells. Peroxidase was localized within the nuclear envelope and endoplasmic reticulum, especially within cells that had lipid inclusions. The degree of peroxidase positivity varied within and among the arteries. In nonlesion regions of the aorta 20% of medial smooth muscle cells was peroxidase positive; the value for coronary artery smooth muscle cells was less. The peroxidase activity within aortic lesions was increased with 44% of intimal smooth muscle cells being positive. Notably, 85-90% of the lipid-containing intimal smooth muscle cells were positive. In contrast, intimal smooth muscle cells in the coronary artery lacked peroxidase reaction product, even in cells containing lipid. We conclude from these studies that aortic lesions contain a cytochemically differentiated subset of lipid-containing, peroxidase-positive smooth muscle cells; but coronary lesions lack a comparable subset of smooth muscle cells.

Animals↗
PubMed2004

Aromatic hydroxamic acids and hydrazides as inhibitors of the peroxidase activity of prostaglandin H2 synthase-2.

The cyclooxygenase activity of the bifunctional enzyme prostaglandin H(2) synthase-2 (PGHS-2) is the target of non-steroidal anti-inflammatory drugs. Inhibition of the peroxidase activity of PGHS has been less studied. Using Soret absorption changes, the binding of aromatic hydroxamic acids to the peroxidase site of PGHS-2 was examined to investigate the structural determinants of inhibition. Typical of mammalian peroxidases, the K(d) for benzhydroxamic acid (42mM) is much greater than that for salicylhydroxamic acid (475microM). Binding of the hydroxamic acid tepoxalin (25microM) resulted in only minor Soret changes. However, tepoxalin is an efficient reducing cosubstrate, indicating that it is an alternative electron donor rather than an inhibitor of the peroxidase activity. Aromatic hydrazides are metabolically activated inhibitors of peroxidases. 2-Naphthoichydrazide (2-NZH) caused the time- and concentration-dependent inhibition of both PGHS-2 peroxidase and cyclooxygenase activities. H(2)O(2) was required for the inactivation of both PGHS-2 activities and indomethacin (which binds at the cyclooxygenase site) did not affect the peroxidase inhibitory potency of 2-NZH. A series of aromatic hydrazides were found to be potent inhibitors of PGHS-2 peroxidase activity with IC(50) values in the 6-100microM range for 13 of the 18 hydrazides examined. Selective inhibition of PGHS-2 over myeloperoxidase and horseradish peroxidase isozyme C was increased by certain ring substitutions. In particular, a chloro group para to the hydrazide moiety increased the PGHS-2 selectivity relative to both myeloperoxidase and horseradish peroxidase isozyme C.

Humans↗
PubMed2004

The class III peroxidase multigenic family in rice and its evolution in land plants.

Plant peroxidases (class III peroxidases, E.C. 1.11.1.7) are secreted glycoproteins known to be involved in the mechanism of cell elongation, in cell wall construction and differentiation, and in the defense against pathogens. They usually form large multigenic families in angiosperms. The recent completion of rice (Oryza sativa japonica c.v. Nipponbare) genome sequencing allowed drawing up the full inventory of the genes encoding class III peroxidases in this plant. We found 138 peroxidase genes distributed among the 12 rice chromosomes. In contrast to several other gene families studied so far, peroxidase genes are twice as numerous in rice as in Arabidopsis. This large number of genes results from various duplication events that were tentatively traced back using a phylogenetic tree based on the alignment of conserved amino acid sequences. We also searched for peroxidase encoding genes in the major phyla of plant kingdom. In addition to gymnosperms and angiosperms, sequences were found in liverworts, mosses and ferns, but not in unicellular green algae. Two rice and one Arabidopsis peroxidase genes appeared to be rather close to the only known sequence from the liverwort Marchantia polymorpha. The possible relationship of these peroxidases with the putative ancestor of peroxidase genes is discussed, as well as the connection between the development of the class III peroxidase multigenic family and the emergence of the first land plants.

Amino Acid Sequence↗
PubMed1986

Selenium independent glutathione peroxidase activity associated with cationic forms of glutathione transferase in human heart.

Glutathione peroxidase activity with both hydrogen peroxide and cumene hydroperoxide was measured in the cytosolic fractions prepared from five human hearts obtained from post-mortem victims. In all the samples the activity with cumene hydroperoxide was higher than that obtained with hydrogen peroxide, suggesting that the selenium-independent glutathione peroxidase could also be present in this tissue. To determine its presence in heart tissue we fractionated the cardiac cytosol fraction on a column of Sephadex G-100 and measured glutathione peroxidase activity with both the substrates. Glutathione transferase activity was measured with 1-chloro-2,4-dinitrobenzene in the fractionated cytosol. The results indicated that a selenium-independent glutathione peroxidase activity was present (about 30% of total activity). Fractionation of the cytosol by gel filtration showed that peroxidase activity co-eluted with glutathione transferase activity. Subsequently the fractions containing glutathione transferase and selenium-independent glutathione peroxidase activity obtained from gel filtration experiments were passed through an affinity column and analyzed by isoelectric focusing. It was found that the selenium-independent glutathione peroxidase copurified with three isoenzymes of glutathione transferase which had a pI of 9.2, 8.9 and 8.6 respectively. In contrast the acidic isoenzymes of glutathione transferase lacked peroxidase activity. It is suggested that the selenium-independent glutathione peroxidase may play an important role in neutralizing oxygen toxicity in heart when the selenium-dependent glutathione peroxidase activity is impaired.

Chromatography, Affinity↗
PubMed1995

Rate enhancement of compound I formation of barley peroxidase by ferulic acid, caffeic acid, and coniferyl alcohol.

Reactions of barley peroxidase 1 were studied using transient-state and steady-state kinetics at pH 3.96, 25 degrees C, and 0.1 M ionic strength, in both the presence and the absence of 1 mM calcium ion. The rate of compound I formation from barley peroxidase 1 and hydrogen peroxide in the absence of reducing substrate is very slow, with or without calcium. When each of the three reducing substrates ferulic acid, caffeic acid, and coniferyl alcohol is added individually, there is a striking enhancement of the rate of compound I formation by a factor of 10-40 depending on the substrate. These unique rate enhancements can be explained by the effect of tight substrate binding to the native enzyme, and they may be indicative of an activating effect of reducing substrate on barley peroxidase 1 under physiological conditions. All steady-state kinetic results can be explained by an initial tight binding of reducing substrate AH to the barley peroxidase, Peroxidase + AH reversible Peroxidase-AH, and substitution of the peroxidase-AH complex for native enzyme in the standard modified ping-pong mechanism for peroxidase reactions [Dunford, H. B. (1991) in Peroxidases in Chemistry and Biology (Everse, J., Everse, K. E., & Grisham, M. B., Eds.) Vol. II, pp 1-24, CRC Press, Boca Raton, FL]. The dissociation constant of barley peroxidase 1 and ferulic acid was 1.4 +/- 0.6 microM as determined by the change in the absorbance at the Soret maximum at the conditions mentioned above.(ABSTRACT TRUNCATED AT 250 WORDS)

Caffeic Acids↗
PubMed1995

Oxidation of yeast iso-1 ferrocytochrome c by yeast cytochrome c peroxidase compounds I and II. Dependence upon ionic strength.

The reduction of cytochrome c peroxidase compound I by excess yeast iso-1 ferrocytochrome c is biphasic. Two pseudo-first-order rate constants can be measured by stopped-flow techniques. The fastest rate process is the reduction of cytochrome c peroxidase compound I to compound II, and the slower process is the reduction of II to the native enzyme. The yeast iso-1 ferrocytochrome c concentration dependence of the reduction of cytochrome c peroxidase compound I to compound II is consistent with a mechanism involving two binding sites for cytochrome c on cytochrome c peroxidase. Electron transfer from cytochrome c bound at the high-affinity binding site to the Fe(IV) site in cytochrome c peroxidase compound I is dependent upon ionic strength, increasing from 15 +/- 6 to 2000 +/- 100 s-1 over the ionic strength range 0.01-0.20 M. The reduction rate of the Fe(IV) site in the 2:1 yeast iso-1 ferrocytochrome c/cytochrome c peroxidase compound I complex is essentially independent of ionic strength with a value of 3800 +/- 300 s-1. The Fe(IV) site in cytochrome c peroxidase compound I is preferentially reduced by yeast ferrocytochrome c between 0.01 and 0.20 M ionic strength while the Trp-191 radical is preferentially reduced above 0.30 M ionic strength. The association rate constant for the binding of yeast iso-1 ferrocytochrome c to cytochrome c peroxidase compound I can be evaluated and varies from a remarkable 1 x 10(10) M-1 s-1 at 0.01 M ionic strength to 1.2 x 10(5) M-1 s-1 at 1.0 M ionic strength. Between 0.01 and 0.20 M ionic strength, the reduction of cytochrome c peroxidase compound II to the native enzyme is anomalous. The reaction is independent of the cytochrome c concentration and directly proportional to the initial cytochrome c peroxidase compound I concentration.

Binding Sites↗
PubMed2003

Protein-based radicals in the catalase-peroxidase of synechocystis PCC6803: a multifrequency EPR investigation of wild-type and variants on the environment of the heme active site.

Catalase-peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 catalase-peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to the heme and spanning the distal and proximal sides were also investigated. In the wild-type enzyme, the EPR signal of the catalases and peroxidases (typical) Compound I intermediate [Fe(IV)=O por.+] was observed. Two protein-based radical intermediates were also detected and identified as a Tyr. and a Trp. . The site of Trp. is proposed to be Trp 106, a residue belonging to the conserved short stretch in catalase-peroxidases and located at a 7-8 A distance to the heme propionate groups. An extensive hydrogen-bonding network on the heme distal side, involving Trp122, His123, Arg119, seven structural waters, the heme 6-propionate group, and Trp106, is proposed to have a key role on the formation of the tryptophanyl radical. We used high-field EPR spectroscopy (95-285 GHz) to resolve the g-anisotropy of the protein-based radicals in Synechocystis catalase-peroxidase. The broad gx component of the HF EPR spectrum of the Tyr. in Synechocystis catalase-peroxidase was consistent with a distributed electropositive protein environment to the tyrosyl radical.

Anisotropy↗