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At least 19 recordsLinked to original sources

PubMed1993

Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. A new family of fungal peroxidases.

Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20-residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8-residue, glycine-rich N-terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N-terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40-45% identical in amino-acid sequence to 11 lignin peroxidases from four fungal species, and 42-43% identical to the two known Mn-peroxidases. Like these white-rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C-terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white-rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less peroxidase polymorphism in C. cinereus strains than observed in plants and white-rot fungi.

Amino Acid Sequence↗

PubMed1995

Multilayer peroxidase-labeled antibody method: comparison with labeled streptavidin-biotin method, avidin-biotin-peroxidase complex method, and peroxidase-antiperoxidase method.

A new, extremely sensitive, "multilayer peroxidase-labeled antibody method" is introduced. This method utilizes peroxidase-labeled antibodies that immuno-react with their antigen, but also can be an antigen to other peroxidase-labeled antibodies. An example of this method is: use rabbit IgG as the first antibody, peroxidase-labeled goat antirabbit IgG as the second antibody, peroxidase-labeled rabbit antigoat IgG as the third antibody, peroxidase-labeled goat antirabbit IgG as the fourth antibody, and peroxidase-labeled rabbit antigoat IgG as the fifth antibody in succession. We compared the sensitivities of this multilayer peroxidase-labeled antibody method with that of commercially available immuno-peroxidase staining kits, i.e., avidin-biotin-complex method and peroxidase-antiperoxidase method, both by immunostaining and enzyme-linked immunosorbent assay. The generated signal and background in immunostained sections were determined by a computer-assisted image analyzer and by enzyme-linked immunosorbent assay spectrophotometrically. It was found that a maximum signal/background ratio was obtained with those sections reacted with the first four antibodies in succession by the multilayer peroxidase-labeled antibody method, and those reacted with the five antibodies in succession by the multilayer peroxidase-labeled antibody method in the enzyme-linked immunosorbent assay. Thus, the multilayer peroxidase-labeled antibody method is more sensitive than the avidin-biotin-complex method and the peroxidase-antiperoxidase method.

Animals↗

PubMed1991

Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency.

Biochemical assays for peroxidase activity do not usually distinguish between different peroxidases. The guaiacol assay, for example, which is one of the most commonly used assays for peroxidase activity, is sensitive to both eosinophil peroxidase (EPO) and the peroxidase of neutrophils, i.e., myeloperoxidase (MPO), thus preventing distinction of the two peroxidases in mixed neutrophil-eosinophil populations. In this paper we describe a simple and sensitive method for selective assays of EPO in EPO-MPO mixtures or mixed populations of neutrophils and eosinophils. The method is based on the peroxidase-mediated oxidation of homovanillic acid (HVA) under appropriate assay conditions in which EPO is still very active in catalyzing the reaction whilst MPO-mediated HVA oxidation is almost undetectable. Optimal assay conditions were as follows: pH 10.5, 10 microM hydrogen peroxide, 0.8 mM HVA and an incubation time of 120 min at 37 degrees C. Under these conditions the assay permits EPO activities as low as 0.025 guaiacol U/ml to be measured even in the presence of 0.175 guaiacol U/ml of MPO. In mixed neutrophil-eosinophil cell suspensions the test permits the detection of as few as 5 X 10(3) eosinophils even in the presence of about 700 X 10(3) neutrophils (eosinophils: neutrophils ratio 1:140) with no appreciable interference by the latter cells. The method described here has been applied to studies of human EPO deficiency and proved to be successful in the identification of individuals with partial EPO deficiency, which is not feasible with non quantitative methods (for example, cytochemistry) or unselective biochemical assay of peroxidase activity.

Eosinophil Peroxidase↗

PubMed1994

Analysis of the optical absorption and magnetic-circular-dichroism spectra of peanut peroxidase: electronic structure of a peroxidase with biochemical properties similar to those of horseradish peroxidase.

The electronic structures of the cationic isoenzyme of peanut peroxidase, horseradish peroxidase (isoenzyme C) and bovine liver catalase are compared through analysis of their optical absorption and magnetic c.d. (m.c.d.) spectral properties. The spectral data for the native resting states and compounds I and II of peanut peroxidase (PeP) are reported. The absorption and m.c.d. data for the native PeP exhibit bands characteristic of the high-spin ferric haem. The absorption spectrum of PeP compound I closely resembles that observed for the HRP compound I species. The m.c.d. data for PeP I clearly identifies that ring oxidation has occurred. One-electron reduction forms the PeP compound II species. The absorption and m.c.d. spectra recorded for PeP II exhibit the well-resolved spectral characteristics previously observed for both HRP compound II and catalase compound II. The spectral data of PeP with HRP and catalase are compared. The data clearly indicate that the m.c.d. spectral patterns of both plant peroxidases (PeP and HRP) are very similar and, therefore, the electronic structures of their resting states, and as well their primary and secondary compounds, must be similar. The m.c.d. data suggest that, while the compound I species of PeP and HRP belong to one electronic class, catalase compound I belongs to a different class. These data emphasize how the ground states of these two classes of oxidized haem, may be characterized as predominantly 2A2u (PeP I and HRP I) or 2A1u (catalase I). Peanut peroxidase is the second plant peroxidase for which the electronic structure of the compound I intermediate has been studied using the m.c.d. technique. The similarities with horseradish peroxidase allow us to suggest that plant peroxidases may operate by the same general mechanism, in spite of the low degree of sequence similarity between their polypeptide chains.

Animals↗

PubMed1999

Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites.

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.

Amino Acid Sequence↗

PubMed2002

A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species.

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.

Animals↗

PubMed2001

Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2.

Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 +/- 0.004 microM(-1) x s(-1) for ATP A2, respectively. The structural origin of the differential reactivity is discussed in relation to glycosylation and amino acid substitutions. The results are of general importance to the use of homologous models and structure determination at low temperatures.

Arabidopsis↗

PubMed2002

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation.

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.

Catalase↗

PubMed1985

Thyroid hormone synthesis and thyroglobulin iodination related to the peroxidase localization of oxidizing equivalents: studies with cytochrome c peroxidase and horseradish peroxidase.

Cytochrome c peroxidase (CcP) and horseradish peroxidase (HRP), when combined with a stoichiometric amount of H2O2, form stable compounds I which are known as FeIV Ro and FeIV o pi + structures, respectively. These compounds were assayed in the catalysis of thyroid hormone synthesis and the iodination reaction. As previously shown for the lactoperoxidase FeIV Ro compound, the CcP FeIV Ro compound was involved in the coupling and not in the iodination reactions. In contrast, the HRP FeIV o pi + compound catalyzed both iodination and hormone formation. The possible role of the different peroxidase-H2O2 compounds in the two sequential reactions, thyroglobulin iodination and thyroid hormone formation, is discussed.

Catalysis↗

PubMed1999

Redesign of cytochrome c peroxidase into a manganese peroxidase: role of tryptophans in peroxidase activity.

Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I'. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1. k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0. 599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism.

Cytochrome-c Peroxidase↗

PubMed1997

Overexpression of cellular glutathione peroxidase does not affect expression of plasma glutathione peroxidase or phospholipid hydroperoxide glutathione peroxidase in mice offered diets adequate or deficient in selenium.

Selenium-dependent cellular glutathione peroxidase (GPX1) overexpressing [GPX1(+)] mice were derived by microinjecting a 5.3-kb cloned entire mouse GPX1 genomic DNA into fertilized eggs. The objective of this study was to determine the effect of GPX1 overexpression and dietary selenium on the expression of selenoperoxidases and the status of lipid peroxidation of these transgenic animals. An experiment with a 2 x 2 factorial arrangement of treatments with 15 GPX1(+) and 15 control mice (2 mo old) was conducted for 8 wk. Ten mice of each group (half males and females) were fed a Se-deficient, Torula yeast basal diet (0.02 mg Se/kg, no supplemental vitamin E) and five mice (three males and two females) were fed the basal diet supplemented with 0.51 mg Se/kg as Na2SeO3. The GPX1(+) mice had greater GPX1 activities (one- to sixfold, P < 0.0001) than the control mice at both levels of dietary selenium in all tissues except for liver, in which such difference (100%, P < 0.05) was observed only in Se-deficient mice. The GPX1 mRNA level in kidney and in lung of the Se-deficient GPX1(+) mice was 81% and 7.5-fold greater (P < 0.003) than the respective control level. Overexpression of GPX1 did not alter phospholipid hydroperoxide glutathione peroxidase (GPX4) activities and mRNA levels or glutathione S-transferase (GST) activities in most of the tissues, plasma glutathione peroxidase (GPX3) activity or plasma Se concentrations. No differences in lipid peroxidation in kidney, lung or intestine were observed between the Se-deficient GPX1(+) and control mice. In conclusion, the overexpression of the GPX1 gene in these mice was tissue specific and did not affect the expression of GPX3, GPX4 or GST and plasma Se levels; dietary Se appeared to affect the GPX1 overexpression at its mRNA level.

Animals↗

PubMed1979

Comparison between peroxidase-conjugated antigen or antibody and peroxidase-anti-peroxidase complex in a postembedding procedure.

The staining efficiency of peroxidase labeled immunoglobulin conjugate, used either as antigen or as antibody, has been compared with that of peroxidase-anti-peroxidase complex (PAP) on ultrathin sections of araldite embedded material. The conjugate gave positive results in a two layer method as well as in a three layer method when used as antibody. No staining was observed when it was used as antigen. The conjugation seemed to impair the antigenic reactivity of immunoglobulin. The conjugate when used as antibody in the three layer method gave approximately the same staining efficiency as PAP.

Animals↗

PubMed1995

Human monoclonal autoantibodies against the immunodominant region on thyroid peroxidase: lack of cross-reactivity with related peroxidases or thyroglobulin and inability to inhibit thyroid peroxidase enzymatic activity.

Thyroid peroxidase (TPO) autoantibodies are heterogeneous and have been classified in terms of whether they cross-react with myeloperoxidase (MPO), lactoperoxidase (LPO), or thyroglobulin (Tg) as well as by whether they inhibit TPO enzymatic activity. Four human monoclonal TPO autoantibodies, generated using combinatorial immunoglobulin gene libraries and expressed as F(ab), have been used to investigate these properties of TPO autoantibodies. The binding of F(ab) WR1.7, TR1.8, TR1.9, and SP1.4 to 125I-labeled recombinant TPO was inhibited 50% by approximately 10(-10) mol/L unlabeled TPO, reflecting the high affinities of these F(ab) for TPO. In contrast, F(ab) binding to TPO was unaffected by human MPO (both native and reduced), bovine LPO, or human Tg at concentrations up to 10(-8) mol/L. Further, TPO enzymatic activity, measured by guiacol oxidation, was unaffected by preincubation with the four F(ab) individually or as a pool (each at 10(-8) mol/L). In conclusion, four human TPO monoclonal autoantibodies do not cross-react with related peroxidases or Tg, nor do they inhibit TPO enzymatic activity. These monoclonal immunoglobulin G class autoantibodies define the immunodominant region on TPO and represent about 85% of TPO autoantibodies in an individual patient's serum. Consequently, our data suggest that TPO autoantibodies that cross-react with MPO, LPO, or Tg, or inhibit TPO enzymatic activity are likely to bind outside the immunodominant region.

Antibodies, Monoclonal↗

PubMed1984

A comparison of the avidin-biotin-peroxidase complex (ABC) and peroxidase-anti-peroxidase (PAP) immunocytochemical techniques for demonstrating Sendai virus infection in fixed tissue specimens.

Mice were infected with Sendai virus and killed 8 days later. Lungs were removed and perfused with ethanol, 10% neutral formalin, Bouin's, B-5, or Zenker's fixatives. Tissues were dehydrated, embedded in paraffin, sectioned and stained for the presence of Sendai virus using the avidin-biotin-peroxidase-complex (ABC) and peroxidase antiperoxidase (PAP) immunocytochemical techniques. Results of these techniques were compared. The ABC technique was more sensitive than the PAP. Sendai antigen was demonstrated by the ABC technique in lung tissue fixed with any fixative, whereas antigen could be demonstrated with consistency only in ethanol-fixed lung by the PAP technique. Trypsin treatment of lung prior to immunoperoxidase treatment failed to enhance staining with either technique and actually caused a decrease in staining in ethanol, B-5 and Zenker's-fixed specimens.

Animals↗

PubMed1989

Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin peroxidase complex (ABC), and peroxidase-labeled avidin-biotin (LAB) methods.

The authors have examined the sensitivity and detection efficiency of the three peroxidase methods that currently have the widest application in diagnostic immunohistochemistry: the peroxidase-antiperoxidase (PAP), the avidin-biotin complex (ABC), and the labeled avidin-biotin (LAB) methods. Sensitivity was evaluated by determining the highest useful dilution of polyclonal antiglucagon antibodies applied to formalin-fixed, paraffin-embedded human pancreas. Detection efficiency was evaluated by tabulation of the total number of positive (three or more positive cells) islets. On direct comparison, the LAB method exceeded the PAP and ABC methods in both sensitivity and detection efficiency, which were essentially equal. Titration of linking antiserum of the PAP method boosted its sensitivity and detection efficiency above that of ABC; the PAP had equal sensitivity to the LAB and exceeded it in detection efficiency. The authors conclude that comparisons of immunohistologic methods are meaningful only if both sensitivity and efficiency are considered along with the unique requirements of any single method.

Avidin↗

PubMed1987

Spinal cord projections from hindlimb muscle nerves in the rat studied by transganglionic transport of horseradish peroxidase, wheat germ agglutinin conjugated horseradish peroxidase, or horseradish peroxidase with dimethylsulfoxide.

The spinal cord projections of four different groups of hindlimb muscle nerve branches--the medial and lateral gastrocnemius nerves, muscle branches of the deep peroneal nerve, muscle branches of the femoral nerve, and a nerve to the hamstring muscles--were studied with transganglionic transport of horseradish peroxidase (HRP) in the rat. The influence of varying the postoperative survival (3, 6, and 10 days) and of using wheat germ agglutinin-HRP conjugate (WGA-HRP), or HRP with dimethylsulfoxide (DMSO) instead of free HRP was studied for the gastrocnemius nerves. After 3 days' survival following application of HRP to the gastrocnemius nerves, fine granular labeling was found mainly in lamina V in L4-5, and coarse granular labeling was found in Clarke's column as far caudally as L2, and in laminae VI and VII predominantly in Th12-L2. After 6 or 10 days' survival, the fine labeling in lamina V was sparse or absent, whereas the coarse labeling appeared to remain or to be only slightly reduced in Clarke's column and in laminae VI and VII. No labeling suggestive of terminals was observed in laminae I-III from the gastrocnemius nerves. Except for sparse labeling in lamina I in some of the cases and some minor differences rostrocaudally, the spinal distribution of labeling was similar to that from the other nerves investigated. The distribution of labeling obtained after application of WGA-HRP or HRP with DMSO to the gastrocnemius nerves was very similar to that obtained with free HRP after 3 days' survival. The results indicate that the spinal cord projections of hindlimb muscle nerves in the rat distribute mainly in the deep part of the dorsal horn and in the intermediate zone. Furthermore, the lack of labeling suggestive of terminals in laminae I-III from the gastrocnemius nerves suggests, in conflict with earlier findings in the cat, that primary afferent fibers from muscles do not necessarily terminate in these laminae in the rat. The results suggest, furthermore, that fine granular labeling found in lamina V represents fine-calibered afferent fibers. Finally, the similar spinal projection patterns of the different muscle nerves investigated suggest either a less developed or an essentially different somatotopic organization for muscle afferents compared to cutaneous afferents, as revealed in earlier studies.

Animals↗

PubMed1985

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase.

The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown. Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared. The three known apoperoxidases consist of 300 +/- 8 amino acid residues. The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively. We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin. The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase. The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described. The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2+-binding sequences in plant peroxidases.

Amino Acid Sequence↗

PubMed1997

From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase.

Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thaliana ascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event.

Amino Acid Sequence↗