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PubMed2002

Analysis and expression of the class III peroxidase large gene family in Arabidopsis thaliana.

Higher plants possess a large set of the classical guaiacol peroxidases (class III peroxidases, E.C. 1.11.1.7). These enzymes have been implicated in a wide array of physiological processes such as H(2)O(2) detoxification, auxin catabolism and lignin biosynthesis and stress response (wounding, pathogen attack, etc.). During the last 10 years, molecular cloning has allowed the isolation and characterization of several genes encoding peroxidases in plants. The achievement of the large scale Arabidopsis genome sequencing, combined with the DNA complementary to RNA (cDNA) expressed sequence tags projects, provided the opportunity to draw up the first comprehensive list of peroxidases in a plant. By screening the available databases, we have identified 73 peroxidase genes throughout the Arabidopsis genome. The evolution of the peroxidase multigene family has been investigated by analyzing the gene structure (intron/exon) in correlation with the phylogenetic relationships between the isoperoxidases. An evolutionary pattern of extensive gene duplications can be inferred and is discussed. Using a cDNA array procedure, the expression pattern of 23 peroxidases was established in the different organs of the plant. All the tested peroxidases were expressed at various levels in roots, while several were also detected in stems, leaves and flowers. The specific functions of these genes remain to be determined.

Arabidopsis↗
PubMed2000

Presence of uterine peroxidase activity in the rat early pregnancy.

Peroxidase has been associated with estrogen action in the uterus. This enzyme plays an important role in the control of hydrogen peroxide levels and in catechol estrogen production. Since the uterus, during early pregnancy, is subjected to estrogen and progesterone regulation, we analyzed the changes of peroxidase activity in relation to receptivity and uterine early response to the embryo. Soluble and microsomal peroxidase activity were determined in the rat uterus during the estrus phase and early pregnancy (days 3 through 6). Soluble peroxidase activity increased significantly (p < 0.01) from day 3 (1.50 +/- 0.24) to day 4 (3.5 +/- 0.3) and 5 (5 +/- 0.5 U/mg protein, mean +/- S.D., n = 6) of pregnancy. During day 6, a significant decrease was noted in both the implantation site and the nonimplantation uterine tissue. Microsomal calcium-extractable peroxidase showed a similar pattern, with lower specific activity than, the soluble peroxidase. During estrus, the uterine tissue showed the highest activity of calcium-extracted peroxidase (8.7 +/- 1.35 U/mg protein), statistically greater when compared with days 3, 4, 5 and 6 of pregnancy. In conclusion, high peroxidase activity was associated with uterine receptivity. The decrease of activity on day 6 might be due to a progesterone-estrogen interaction, and consequently, hydrogen peroxide can be utilized for hydroxile production by means of the Fenton reaction. Lipoperoxidation may be necessary for changes in membrane fluidity for embryo attachment to endometrial epithelium.

Animals↗
PubMed1987

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme.

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗
PubMed1989

Oxidation-reduction potentials and ionization states of extracellular peroxidases from the lignin-degrading fungus Phanerochaete chrysosporium.

The oxidation-reduction potentials of lignin peroxidase isozymes H1, H2, H8, and H10 as well as the Mn-dependent peroxidase isozymes H3 and H4 are reported. The potentiometric titrations involving the ferrous and ferric states of the enzyme had Nernst plots indicating single-electron transfer. The Em7 values of lignin peroxidase isozymes H1, H2, H8, and H10 are -142, -135, -137, and -127 mV versus standard hydrogen electrode, respectively. The Em7 values for the Mn-dependent peroxidase isozymes H3 and H4 are -88 and -93 mV versus standard hydrogen electrode, respectively. The midpoint potential of H1, H8, and H4 remained unchanged in the presence of their respective substrates, veratryl alcohol and Mn(II). The midpoint potential between the ferric and ferrous forms of isozymes H1 and H4 exhibited a pH-dependent change between pH 3.5 and pH 6.5. These results indicate that the reductive half-reaction of the enzymes is the following: ferric peroxidase + le- + H+----ferrous peroxidase. Above pH 6.5, the effect of pH on the midpoint potential is diminished and indicates that an ionization with an apparent pKa equal to approximately 6.6-6.7 occurs in the reduced form of the enzymes. A heme-linked ionization group in the ferrous form of the enzymes was confirmed by studying the effect of pH on the absorption spectra of isozymes H1 and H4. These spectrophotometric pH titration experiments confirmed the electrochemical results indicating pKa values of 6.59 and 6.69 for reduced isozymes H1 and H4, respectively. These results indicate the presence of a heme-linked ionization of an amino acid in the reduced form of the lignin peroxidase isozymes similar to that of other plant peroxidases.

Basidiomycota↗
PubMed1996

Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking.

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.

Animals↗
PubMed1997

Role of calcium in maintaining the heme environment of manganese peroxidase.

We previously demonstrated that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was very susceptible to thermal inactivation due to the loss of calcium from the enzyme [Sutherland & Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. The structural changes that occur during thermal inactivation and the release of calcium from manganese peroxidase have now been characterized. Thermal inactivation caused distinct alterations in the heme environment and slight changes in the overall protein structure, both of which were reversed upon reactivation of the enzyme with calcium. The absorption spectrum of inactivated manganese peroxidase was similar to that of low-spin ferric heme proteins, indicating that a sixth ligand had bound to the distal side of the heme iron. Consistent with disruption of the distal heme environment, thermally inactivated manganese peroxidase did not react with hydrogen peroxide to form compound I. The inactive enzyme exhibited a pH-dependent absorption transition with a pKa of 5.7. Studies involving imidazole indicated that the sixth ligand may be a distal histidine. Low-temperature electron paramagnetic resonance spectroscopy confirmed that the heme iron of the inactivated form of manganese peroxidase was predominantly in a low-spin state. The near-ultraviolet/visible circular dichroism spectrum also supported the proposed formation of a highly symmetric bis(imidazole) heme complex upon thermal inactivation of the enzyme. A recombinant manganese peroxidase, in which the distal calcium binding site was altered such that calcium binding would be minimized, was also characterized. This enzyme, D47A, had the same catalytic and spectroscopic properties and calcium content as thermally inactivated manganese peroxidase. Therefore, the inactivation and structural changes that occurred during thermal incubation of manganese peroxidase could be explained by the loss of the distal calcium.

Basidiomycota↗
PubMed1997

Catalytic activities and structural properties of horseradish peroxidase distal His42 --> Glu or Gln mutant.

The distal histidine (His) is highly conserved in peroxidases and has been considered to play a major role as a general acid-base catalyst for peroxidase reaction cycle. Recently, however, the X-ray structure of chloroperoxidase from the marine fungus Caldariomyces fumago has revealed that a glutamic acid is located at the position where most of the peroxidase has a histidine residue, suggesting that the carboxyl group in the glutamic acid (Glu) can also assist cleavage of an O-O bond in peroxides [Sundaramoorthy, M., Terner, J., & Poulos, T. L. (1995) Structure 3, 1367-1377]. In order to investigate catalytic roles of the glutamic acid at the distal cavity, two horseradish peroxidase mutants were prepared, in which the distal His42 has been replaced by Glu (H42E) or Gln (H42Q). The formation rate of compound I in the H42E mutant was significantly greater than that for the H42Q mutant, indicating that the distal Glu can play a role as a general acid-base catalyst. However, the peroxidase activity of the H42E mutant was still lower, compared to that for native enzyme. On the basis of the CD, resonance Raman, and EPR spectra, it was suggested that the basicity of the distal Glu is lower than that of the distal His and the position of the distal Glu is not fixed at the optimal position as a catalytic amino acid residue, although no prominent structural changes around heme environment were detected. The less basicity and improper positioning of the distal Glu would destabilize the heme-H2O2-distal Glu ternary intermediate for the peroxidase reaction. Another characteristic feature in the mutants was the enhancement of the peroxygenase activity. Since the peroxygenase activity was remarkably enhanced in the H42E mutant, the distal Glu is also crucial to facilitate the peroxygenase activity as well as the enlarged distal cavity caused by the amino acid substitution. These observations indicate that the distal amino acid residue is essential for function of peroxidases and subtle conformational changes around the distal cavity would control the catalytic reactions in peroxidase.

Catalysis↗
PubMed2001

Induction of soluble and cell wall peroxidases by aphid infestation in barley.

Peroxidase enzymes have been found in soluble, ionically bound, and covalently bound forms and have been implicated in several physiological processes in plants. This paper investigates the effect of aphid infestation on soluble and bound-cell wall peroxidase activity and bound-cell wall isoform changes of barley plants. Peroxidase activity was measured in control plants and plants infested with the aphid Schizaphis graminum (Rondani). The activity of soluble peroxidases increased with time of infestation, older plants being more affected than younger ones. The increase in bound-cell wall peroxidase activity as a function of age was higher in infested than in control plants, being higher in ionically bound than in covalently bound peroxidases. When the aphids were removed from plants, the activities of both types of peroxidases decreased to control levels. Isoelectrofocusing analyses of the ionically bound peroxidases showed changes in the isoform pattern. A new isoform was induced by infestation. The activities of all covalently bound isoforms increased after infestation. The physiological implications of these changes are discussed.

Animals↗
PubMed1971

Complex-formation between cytochrome c and cytochrome c peroxidase. Equilibrium and titration studies.

1. Physical studies of complex-formation between cytochrome c and yeast peroxidase are consistent with kinetic predictions that these complexes participate in the catalytic activity of yeast peroxidase towards ferrocytochrome c. Enzyme-ferricytochrome c complexes have been detected both by the analytical ultracentrifuge and by column chromatography, whereas an enzyme-ferrocytochrome c complex was demonstrated by column chromatography. Estimated binding constants obtained from chromatographic experiments were similar to the measured kinetic values. 2. The physicochemical study of the enzyme-ferricytochrome c complex, and an analysis of its spectrum and reactivity, suggest that the conformation and reactivity of neither cytochrome c nor yeast peroxidase are grossly modified in the complex. 3. The peroxide compound of yeast cytochrome c peroxidase was found to have two oxidizing equivalents accessible to cytochrome c but only one readily accessible to ferrocyanide. Several types of peroxide compound, differing in available oxidizing equivalents and in reactivity with cytochrome c, seem to be formed by stoicheiometric amounts of hydrogen peroxide. 4. Fluoride combines not only with free yeast peroxidase but also with peroxidase-peroxide and accelerates the decomposition of the latter compound. The ligand-catalysed decomposition provides evidence for one-electron reduction pathways in yeast peroxidase, and the reversible binding of fluoride casts doubt upon the concept that the peroxidase-peroxide intermediate is any form of peroxide complex. 5. A mechanism for cytochrome c oxidation is proposed involving the successive reaction of two reversibly bound molecules of cytochrome c with oxidizing equivalents associated with the enzyme protein.

Chromatography↗
PubMed2003

Histidine 386 and its role in cyclooxygenase and peroxidase catalysis by prostaglandin-endoperoxide H synthases.

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG2 to PGH2. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr385 to a tyrosyl radical within the cyclooxygenase site. His386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr385, which protrudes into the cyclooxygenase site, and His388, the proximal ligand of the peroxidase heme. When His386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His386 helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N-delta of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-delta of His386 and the backbone carbonyl oxygens of Asn382 and Gln383. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly526 and Ser530 and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.

Animals↗
PubMed2002

A retrospective look at the cationic peanut peroxidase structure.

The cationic peanut peroxidase has been studied in detail, not only with regard to its peptide structure, but also to the sites and role of the three moieties linked to it. Peanut peroxidase lends itself well to a close examination as a potential example for other plant peroxidase studies. It was the first plant peroxidase for which a 3-D structure was derived from crystals, with the glycans intact. Subsequent analysis of peroxidases structures from other plants have not shown great differences to that of the peanut peroxidase. As the period of proteomics follows on the era of genomics, the study of glycans has been brought back into focus. With the potential use of peroxidase as a polymerization agent for industry, there are some aspects of the overall structure that should be kept in mind for successful use of this enzyme. A variety of techniques are now available to assay for these structures/moieties and their roles. Peanut peroxidase data are reviewed in that light, as well as defining some true terms for isozymes. Because a high return of the enzyme in a pure form has been obtained from cultured cells in suspension culture, a brief review of this is also offered.

Arachis↗
PubMed1970

The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver.

Endogenous peroxidase activity has been demonstrated in sections of rat liver fixed briefly by glutaraldehyde perfusion and incubated in Graham and Karnovsky's medium for cytochemical demonstration of peroxidase activity (29). In 25-40% of sinusoidal cells, an electron-opaque reaction product is localized in segments of the endoplasmic reticulum, including the perinuclear cisternae, a few Golgi vesicles and saccules and in some large membrane-bounded granules. This staining is abolished after prolonged fixation or boiling of tissue sections in glutaraldehyde, and in the absence of H(2)O(2) or DAB from the incubation medium. Furthermore, the reaction is inhibited completely by sodium azide and high concentrations of H(2)O(2), and partially by KCN and aminotriazole. Among the different cells in hepatic sinusoids, the nonphagocytic "fat-storing" cells (39) are always peroxidase negative, whereas the lining cells in process of erythrophagocytosis are consistently peroxidase positive. The possible biological significance of endogenous peroxidase in Kupffer cells is discussed. In addition, the uptake of exogenous horseradish peroxidase by Kupffer cells has been investigated. The exogenous tracer protein, which in contrast to endogenous peroxidase of Kupffer cells is not inhibited by prolonged aldehyde fixation, is taken up by micropinocytosis and remains confined to the lysosomal system of Kupffer cells. The significance of these observations in respect to some recent studies suggesting localization of exogenous peroxidases in the endoplasmic reticulum of Kupffer cells and peritoneal macrophages (22, 23) is briefly discussed.

Aldehydes↗
PubMed1998

Proximal and distal histidines in thyroid peroxidase: relation to the alternatively spliced form, TPO-2.

The distal and proximal histidines in thyroid peroxidase (TPO), located by amino acid sequence alignment with their known counterparts in myeloperoxidase, are His 239 and His 494, respectively. These histidines lie outside the 57 amino acid peptide (residues 533-589) that is absent in the alternatively spliced form, TPO-2. However, asparagine 579, which very likely forms a stabilizing hydrogen bond with the proximal histidine in TPO, lies within the missing peptide region. The absence of Asn 579 from TPO-2 may be at least partially responsible for the reported lack of activity of this form of the enzyme. Formation of TPO compound I may also depend on Arg 396, based on analogy with the catalytic mechanism previously proposed for the more widely studied plant and fungal peroxidases. A multiple sequence alignment prepared with five mammalian and five invertebrate peroxidases shows complete conservation of Arg 396, as well as residues corresponding to His 239, His 494, and Asn 579 in TPO. The animal peroxidases comprise a family of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary, and tertiary structure, yet share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relation between animal and nonanimal peroxidases may represent an example of convergent evolution to a common enzymatic mechanism.

Alternative Splicing↗
PubMed1987

A case of lowered eosinophilic leucocyte peroxidase activity found unexpectedly using an automated haematology analyser (the Technicon H6000 system)

The Technicon H6000 system simultaneously determines blood cell and leucocyte differential counts automatically using cytochemistry and cell size measurements. Part of the information provided by this instrument is a plot of peroxidase staining intensity against leucocyte cell size called 'the leucocyte peroxidase distribution display'. Recently, an abnormal leucocyte peroxidase distribution display was observed, on a sample from a post-operative lung cancer patient, in which eosinophilic leucocytes did not react with peroxidase. Neutrophils reacted in a normal way with this sample. The eosinophils were further examined by electron microscopy and manual peroxidase staining. Some peroxidase positive granules were observed in the eosinophilic leucocytes. The abnormal leucocyte peroxidase distribution display was found to be due to a separate group of eosinophilic leucocytes with lowered peroxidase activity.

Blood Cell Count↗
PubMed2004

Role of peroxidase inhibition by insulin in the bovine thyroid cell proliferation mechanism.

Monolayer primary cultures of thyroid cells produce, in the presence of insulin, a cytosolic inhibitor of thyroid peroxidase (TPO), lacto peroxidase (LPO), horseradish peroxidase (HRPO) and glutathione peroxidase (GPX). The inhibitor, localized in the cytosol, is thermostable and hydrophylic. Its molecular mass is less than 2 kDa. The inhibitory activity, resistant to proteolytic and nucleolytic enzymes, disappears with sodium metaperiodate treatment, as an oxidant of carbohydrates, supporting its oligosaccharide structure. The presence of inositol, mannose, glucose, the specific inhibition of cyclic AMP-dependent protein kinase and the disappearance of peroxidase inhibition by alkaline phosphatase and alpha-mannosidase in purified samples confirms its chemical structure as inositol phosphoglycan-like. Purification by anionic interchange shows that the peroxidase inhibitor elutes like the two subtypes of inositol phosphoglycans (IPG)P and A, characterized as signal transducers of insulin action. Insulin significantly increases the concentration of the peroxidase inhibitor in a thyroid cell culture at 48 h. The addition of both isolated substances to a primary thyroid culture produces, after 30 min, a significant increase in hydrogen peroxide (H2O2) concentration in the medium, concomitantly with the disappearance of the GPX activity in the same conditions. The presence of insulin or anyone of both products, during 48 h, induces cell proliferation of the thyroid cell culture. In conclusion, insulin stimulates thyroid cell division through the effect of a peroxidase inhibitor, as its second messenger. The inhibition of GPX by its action positively modulates the H2O2 level, which would produce, as was demonstrated by other authors, the signal for cell proliferation.

Animals↗
PubMed1977

Relationship between antigenic stimulation and increased splenic peroxidase levels during the immune response.

Immunization of mice with either soluble or particulate antigens induced a marked increase in peroxidase activity of spleen cell homogenates. Animals immunized with sheep erythrocytes showed maximum splenic peroxidase activity at 2 days. By the 4th day after immunization, when the hemolytic antibody plaque-forming cell response was maximal, peroxidase activity returned to normal levels. Increased splenic peroxidase activity also occurred in mice immunized with rabbit erythrocytes, as well as with rabbit serum or bovine serum albumin. No change in splenic peroxidase activity occurred in mice injected with syngeneic mouse erythrocytes or serum. Both glass-adherent spleen cell populations, morphologically consisting of 90 to 95% macrophages, as well as nonadherent cells, consisting of more than 90% lymphocytes by the same criteria, showed peroxidase activity. Immunization of mice with sheep erythrocytes resulted in an intracellular redistribution of the peroxidase activity among several distinct subcellular fractions prepared by differential centrifugation. Maximum redistribution occurred with granule-associated enzyme activity. A possible relationship between peroxidase activity with functional activity of lymphocytes and macrophages during humor immune responses seemed likely.

Animals↗
PubMed1986

Catalases, peroxidases, and superoxide dismutases in Mycobacterium leprae and other mycobacteria studied by crossed immunoelectrophoresis and polyacrylamide gel electrophoresis.

The five mycobacteria Mycobacterium lepraemurium, M. leprae, M. bovis BCG, M. smegmatis, and M. intracellulare were studied. Catalase and peroxidase activities were demonstrated in polyacrylamide and crossed immunoelectrophoresis gels for M. lepraemurium, M. intracellulare, and BCG, but not for M. leprae. Peroxidase and catalase activities were associated with the same precipitate line in crossed immunoelectrophoresis for M. lepraemurium, M. intracellulare, and BCG, showing that in these mycobacteria the two enzyme activities resided in the same molecule. M. smegmatis peroxidase and catalase activities were closely associated on polyacrylamide gel electrophoresis, but on the crossed immunoelectrophoresis catalase and peroxidase activities were associated with two different precipitate lines. Catalases without peroxidase activity were demonstrated in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in M. intracellulare and M. smegmatis. The catalase without peroxidase activity in M. intracellulare was heat resistant and therefore classified as an m-catalase. In M. smegmatis the catalase without peroxidase activity was only partially heat resistant. All of the catalases with peroxidase activity were heat-sensitive t-catalases. Superoxide dismutase activity in the crossed immunoelectrophoresis was associated with the M. leprae antigen no. 4 and with cross-reacting antigens in the other mycobacteria studied. Several superoxide dismutases were demonstrated in Mycobacterium duvalii. They were antigenically different from the other superoxide dismutases in this study, as shown by lack of reactivity with a monospecific antibody to M. lepraemurium superoxide dismutase. Molecular weights were estimated for all the enzymes in this study by sodium dodecyl sulfate-polyacrylamide gels.

Catalase↗
PubMed1989

Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.

Amino Acid Sequence↗