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PubMed1984

Application of peroxidase-antiperoxidase (PAP) staining for detection and localization of dengue-2 antigen. I. In an endogenous peroxidase containing cell systems.

The unlabelled immunoperoxidase, peroxidase-anti-peroxidase (PAP), technique was used to detect dengue type-2 viral antigen in several cell systems including the endogenous peroxidase containing cells. These cells are the mosquito cell line (C6/36), continuous cell line of rhesus monkey kidney (LLC-MK2), human monocyte culture both cell suspension and monolayer, and human peripheral blood leukocytes. All of these specimens gave the same results that dengue-2 viral antigen presented in cytoplasm only and the patterns of marker presentation in positive cells varied depending on the duration after infection. The sensitivity of this method is extremely high since it can detect dengue-2 antigen after its attachment on mosquito cells (15 min) as seen in experiments with mosquito cell line, C6/36. False positive was not observed in all cell systems tested.

Animals↗
PubMed1998

Central projections of nerves innervating the rabbit maxillary sinus localized using wheat germ agglutinin-horseradish peroxidase or choleragenoid-horseradish peroxidase.

Central projections of nerves innervating the rabbit maxillary sinus were localized by using wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or choleragenoid-horseradish peroxidase (B-HRP). Tracer was placed into the left maxillary sinus; rabbits were killed 3 or 5 days later, and histochemical localization of transported WGA-HRP or B-HRP was performed. Labeled cell bodies (437-545/animal) were seen in the ipsilateral trigeminal ganglion. Very few labeled cell bodies (zero to three/animal) were observed in the contralateral ganglion. The area of cell bodies labeled by WGA-HRP appeared similar to the area of cell bodies labeled by B-HRP. Transganglionic projections from either tracer were localized to lamina II of the ipsilateral subnucleus caudalis. In addition, WGA-HRP labeling was occasionally observed in lamina I. No labeling was present in other areas of the brainstem. In contrast to the above results, other studies have demonstrated that B-HRP produces terminal-like labeling in deeper layers of the gray matter. We injected B-HRP into the infraorbital nerve and sciatic nerve, which are known to contain projections to deep layers of the gray matter. Labeling was observed in the deep layers of the medullary or spinal dorsal horn 5 days later, suggesting that nerves innervating the sinus only project to superficial laminae. These results suggest that neurons in superficial laminae of the subnucleus caudalis may be important for the reflex initiation of the increased glandular secretions in the maxillary sinus during sinusitis.

Afferent Pathways↗
PubMed2000

Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats.

Severe steroidogenic and spermatogenic alterations are reported in association with diabetic manifestations in humans and experimental animals. This study was planned to determine whether oxidative stress is involved in diabetes-induced alterations in the testes. Diabetes was induced in male rats by injection of 50 mg/kg of streptozotocin (STZ). Ten weeks after injection of STZ, levels of selenium and activities of selenium dependent-glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were measured in rat testis. Lipid and protein oxidations were evaluated as measurements of testis malondialdehyde (MDA) and protein carbonyl levels, respectively. Testis sulfydryl (SH) levels were also determined. The control levels of GPx and PHGPx activities were found to be 46.5 +/- 6.2 and 108.8 +/- 19.8 nmol GSH/mg protein/min, respectively. Diabetes caused an increase in testis GPx (65.0 +/- 21.1) and PHGPx (155.9 +/- 43.1) activities but did not affect the levels of selenium or SH. However, the testis MDA and protein carbonyl levels as markers of lipid and protein oxidation, respectively, did not increase in the diabetic group. Aminoguanidine (AG) treatment of diabetic rats returned the testis PHGPx activity (136.5 +/- 24.9) to the control level but did not change the value of GPx activity (69.2 +/- 17.4) compared with diabetic group. MDA and protein carbonyl levels in testis were not affected by AG treatment of diabetic rats, but interestingly AG caused SH levels to increase. The results indicate that reactive oxygen radicals were not involved in possible testicular complications of diabetes because diabetes-induced activations of GPx and PHGPx provided protection against oxidative stress, which was reported to be related to some diabetic complications.

Animals↗
PubMed1986

Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system.

A number of methods for demonstration of peroxidase activity have been tested on immunocytochemical nitrocellulose models. By applying a well-characterized primary antibody and Sternberger's peroxidase-antiperoxidase technique, the sensitivity of various protocols has been evaluated. Best results were obtained with diaminobenzidine as chromogen, especially in conjunction with heavy metal salts, either added directly to the medium or used as "toners" of the end-product. Use of silver intensification of the diaminobenzidine/metal end-product increased sensitivity further.

3,3'-Diaminobenzidine↗
PubMed2003

Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase.

Cytochrome c peroxidase (C cP) variants with an engineered Mn(II) binding site, including MnC cP [C cP(MI, G41E, V45E, H181D)], MnC cP(W191F), and MnC cP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200-700 cm(-1) and 1300-1650 cm(-1) regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of C cP(MI), the recombinant yeast C cP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the nu(3) mode indicate that a pure five-coordinate heme iron exists in C cP(MI) whereas a six-coordinate low-spin iron is the dominant species in the C cP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants C cP(MI, W191F) and C cP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H(2)O(2) and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnC cP(W191F, W51F).

Amino Acid Substitution↗
PubMed1984

Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays.

The periodate-mediated conjugation of horseradish peroxidase to antibody is one of the most popular methods to prepare conjugates for enzyme immunoassays of antigens or corresponding antibodies. A very simple method to obtain peroxidase, which is both about five times cheaper than the rather expensive commercial preparations and has a significant higher activity, is reported. Moreover, the conjugation method was critically investigated and considerably simplified. Conjugates thus obtained are about three times more active than the best obtained with the original method.

Antigen-Antibody Complex↗
PubMed1988

Peroxidase activity at CNS nodes of Ranvier and in initial axon segments of lumbosacral alpha-motoneurons after intramuscular administration of horseradish peroxidase.

The occurrence of peroxidase activity in central (CNS) and peripheral nervous system (PNS) parts of alpha-motor axons was studied by light and electron microscopy in adult cats after injection of horseradish peroxidase (HRP) into the medial gastrocnemius muscle. The intrafunicular parts of the axons were virtually free of HRP-positive bodies except at a few nodes of Ranvier. Most of these nodes were weakly HRP-positive and contained, irrespective of a survival time between 25 and 48 h, only a few HRP-positive bodies randomly scattered in the nodal axoplasm. In contrast to this and as described elsewhere (J. Neurocytol., 15 [1986] 253-260), the nodal regions of alpha-motor axons at the level of the ventral root showed strong and characteristic accumulations of HRP-activity. The initial axon segments and adjoining axonal parts contained many HRP-positive bodies. We conclude that the CNS and the PNS parts of an alpha-motor axon differ with regard to the way nodal regions interact with retrogradely transported HRP. Possible mechanisms behind this difference are discussed.

Animals↗
PubMed1996

Selective control of cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNA stability by selenium supply.

Selenium depletion of H4 hepatoma cells reduced cytosolic glutathione peroxidase (cGSH-Px) mRNA abundance but had no effect on phospholipid hydroperoxide glutathione peroxidase (PHGSH-Px) mRNA abundance. Actinomycin D chase experiments showed that selenium depletion had no effect on the stability of PHGSH-Px mRNA but decreased the stability of cGSH-Px mRNA. In Se-replete cells puromycin decreased the stability of both cGSH-Px and PHGSH-Px mRNAs. The results suggest that when selenium supply is limiting PHGSH-Px mRNA translation is maintained more than that of cGSH-Px mRNA, and thus more cGSH-Px mRNA is released from polysomes and degraded.

Animals↗
PubMed1997

Role of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase in the reduction of lysophospholipid hydroperoxides.

1-linoleoyl lysophosphatidylcholine hydroperoxide is a substrate of GSH peroxidase (GPx) both purified from bovine erythrocytes and nonpurified from rat liver. The initial reaction rate for bovine erythrocyte GPx with 1-linoleoyl lysophosphatidylcholine hydroperoxide is about 76 and 95% of the reaction rate for hydrogen peroxide and linoleic acid hydroperoxide respectively. For rat liver GPx these initial reaction rates are about 66 and 75%, respectively. The rate constants for the reaction of GPx with 1-linoleoyl lysophosphatidylcholine hydroperoxide were calculated to be approximately 3 x 10(7) M-1s-1 and approximately 2 x 10(6) M-1s-1 for the bovine erythrocyte and the rat liver enzymes, respectively. By using kinetic models of lipid peroxidation we found by simulation that: (1) the main source of lysophospholipid hydroperoxides in vivo is the peroxidation of lysophospholipids, both in mitochondrial inner membranes and in endoplasmic reticulum; (2) a specialized enzyme able to reduce directly lysophospholipid hydroperoxides is important for the reduction of these hydroperoxides, because the detoxification of these species mediated by the action of acyl ester bond cleaving enzymes is not efficient; (3) the reduction through GPx predominates over phospholipid hydroperoxide glutathione peroxidase (PHGPx) in mitochondrial inner membranes and in the cytosolic phase of the endoplasmic reticulum; (4) in the luminal phase of endoplasmic reticulum PHGPx is predominant.

Animals↗
PubMed1999

Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.

The heme active site structure of an engineered cytochrome c peroxidase [MnCcP; see Yeung, B. K., et al. (1997) Chem. Biol. 4, 215-221] that closely mimics manganese peroxidase (MnP) has been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CepsilonH shift of the proximal histidine and (15)N shift of the bound C(15)N(-) suggest a weaker heme Fe(III)-N(His) bond in MnCcP compared to WtCcP. Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)-N(His) bond strength of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely.

Arginine↗
PubMed1999

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.

Amino Acid Substitution↗
PubMed2005

Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain.

Haem-containing enzymes (peroxidase and catalase) are widely distributed among prokaryotes and eukaryotes and play a vital role in H2O2 detoxification. But, to date, no haem-containing enzymatic defence against toxic H2O2 has been discovered in Leishmania species. We cloned, expressed and purified an unusual plant-like APX (ascorbate peroxidase) from Leishmania major (LmAPX) and characterized its catalytic parameters under steady-state conditions. Examination of its protein sequence indicated approx. 30-60% identity with other APXs. The N-terminal extension of LmAPX is characterized by a charged region followed by a stretch of 22 amino acids containing a transmembrane domain. To understand how the transmembrane domain influences the structure-function of LmAPX, we generated, purified and extensively characterized a variant that lacked the transmembrane domain. Eliminating the transmembrane domain had no impact on substrate-binding affinity but slowed down ascorbate oxidation and increased resistance to H2O2-dependent inactivation in the absence of electron donor by 480-fold. Spectral studies show that H2O2 can quickly oxidize the native enzyme to compound (II), which subsequently is reduced back to the native enzyme by an electron donor. In contrast, ascorbate-free transmembrane domain-containing enzyme did not react with H2O2, as revealed by the absence of compound (II) formation. Our findings suggest that the single copy LmAPX gene may play an important role in detoxification of H2O2 that is generated by endogenous processes and as a result of external influences such as the oxidative burst of infected host macrophages or during drug metabolism by Leishmania.

Amino Acid Sequence↗
PubMed1996

Role of the 3' untranslated region in the regulation of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase gene expression by selenium supply.

Selenium is an essential nutrient and synthesis of selenoproteins is affected by limited selenium supply. During selenium deficiency there is a differential regulation of selenoprotein synthesis and gene expression; for example, there is a decrease in abundance of mRNA for cytosolic glutathione peroxidase (cGSH-Px) and a preservation of mRNA for phospholipid-hydroperoxide glutathione peroxidase (PHGSH-Px). This difference is not due to an alteration in the rate of transcription but might reflect differences in translation. The aim of the present work was to assess the role of cGSH-Px and PHGSH-Px 3' untranslated regions (UTRs) in the regulation of selenoprotein mRNA stability and translation by using H4-II-E-C3 cells transfected with different constructs containing a type I iodothyronine deiodinase-coding region linked to different selenoprotein mRNA 3' UTRs. Translational efficiency results showed that the efficiency of the 3' UTRs in permitting selenocysteine incorporation is similar in selenium-replete conditions but, when selenium is limiting, the 3' UTR of cGSH-Px is less efficient than the 3' UTR of PHGSH-Px. The results suggest that the 3' UTR of these selenoprotein mRNA species influences their extent of translation when selenium levels are low. The different sensitivity of the 3' UTRs to selenium deficiency can explain the differential effect that selenium deficiency has on cGSH-Px and PHGSH-Px activity and mRNA levels, stability and translation. This might be partly responsible for channelling selenium for synthesis of PHGSH-Px rather than cGSH-Px.

Animals↗
PubMed2004

Native specific activity of glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo- and hypomotile human sperm samples.

Glutathione-dependent selenoenzymes in human spermatozoa are responsible for a generalized protection against reactive oxygen species (ROS) as well as some other metabolic and structural regulation during spermiogenesis and sperm cell maturation. Glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (GPx-4 or PHGPx) and glutathione reductase (GR) native specific activities have been studied in human Percoll-purified spermatozoa from healthy fertile subjects and asthenozoospermic patients. The mean values obtained for the three enzymes in normal specimens are 1.52 +/- 0.90 mU/10(6) sperm cells (PHGPx), 4.26 +/- 1.73 mU/10(6) sperm cells (GPx-1) and 1.95 mU/10(6) sperm cells (GR). No statistically significant differences for any of the three enzymes were encountered between these values and those of asthenozoospermic patients. These results are discussed and compared with recent literature data on both rescued and native PHGPx specific activity in human spermatozoa, as well as with data obtained for GPx in human seminal plasma.

Adult↗
PubMed2002

Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities.

This study investigated the enzymatic function of two putative plant GPXs, GPXle1 from Lycopersicon esculentum and GPXha2 from Helianthus annuus, which show sequence identities with the mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPX). Both purified recombinant proteins expressed in Escherichia coli show PHGPX activity by reducing alkyl, fatty acid and phospholipid hydroperoxides but not hydrogen peroxide in the presence of glutathione. Interestingly, both recombinant GPXle1 and GPXha2 proteins also reduce alkyl, fatty acid and phospholipid hydroperoxides as well as hydrogen peroxide using thioredoxin as reducing substrate. Moreover, thioredoxin peroxidase (TPX) activities were found to be higher than PHGPX activities in terms of efficiency and substrate affinities, as revealed by their respective Vmax and Km values. We therefore conclude that these two plant GPX-like proteins are antioxidant enzymes showing PHGPX and TPX activities.

Antioxidants↗
PubMed1999

Catalase-peroxidases in cyanobacteria--similarities and differences to ascorbate peroxidases.

Cyanobacteria (blue-green algae) are oxygenic phototrophic bacteria carrying out plant-type photosynthesis. The only hydrogen peroxide scavenging enzymes in at least two unicellular species have been demonstrated to be bifunctional cytosolic catalase-peroxidases (CatPXs) having considerable homology at the active site with plant ascorbate peroxidases (APXs). In this paper we examined optical and kinetic properties of CatPXs from the cyanobacteria Anacystis nidulans and Synechocystis PCC 6803 and discuss similarities and differences to plant APXs. Both CatPXs and APX showed similar spectra of the ferric enzyme, the redox intermediate Compound I and the cyanide complex, whereas the spectrum of CatPX Compound II had characteristics reminiscent of the spectrum of the native enzyme. Both steady-state and multi-mixing transient-state kinetic studies were performed in order to characterize the kinetic behaviour of CatPXs. Bimolecular rate constants of both formation and reduction of a CatPX Compound I are presented. Because of its intrinsic high catalase activity (which cannot be found in APXs), the rate constants for Compound I formation were measured with peroxoacetic acid and are shown to be 5.9 x 10(4) M(-1) s(-1) for CatPX from A. nidulans and 8.7 x 10(3) M(-1) s(-1) for the Synechocystis enzyme. Compared with o-dianisidine (2.7-6.7 x 10(6) M(-1) s(-1)) and pyrogallol (8.6 x 10(4)-1.6 x 10(5) M(-1) s(-1)), the rate constant for Compound I reduction by ascorbate was extremely low (5.4 x 10(3) M(-1) s(-1) at pH 7.0 and 15 degrees C), in marked contrast to the behaviour of APXs.

Ascorbate Peroxidases↗
PubMed1995

Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium.

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) and classical glutathione peroxidase (GPX1) are encoded by separate genes with only about 40% amino acid and nucleic acid sequence identity. To determine the response of tissue PHGPX expression to dietary Se level and to compare these responses with those for GPX1, weanling male rats were fed amino acid diets containing from 2 (-Se) to 130 (+Se) microgram Se/kg diet or a torula diet containing 5 and 190 micrograms Se/kg diet as Na2SeO3 for 28 d. Tissues were analyzed for PHGPX and GPX1 activity and mRNA. There was no effect of Se on growth. In -Se rats, GPX1 activity was reduced to 1% in liver and 4-9% in heart, kidney and lung compared with +Se rats; PHGPX activity was reduced only to 25-50% in these four tissues. The Se response curves indicated that the dietary Se requirement to reach plateau liver PHGPX activity was half that required for plateau GPX activity. In -Se rats, liver and heart GPX1 mRNA levels were reduced to 6 and 12%, respectively, whereas PHGPX mRNA was not significantly affected by Se deficiency. Notably, 65 micrograms Se/kg diet resulted in plateau liver GPX1 mRNA levels but not plateau GPX activity. Testis had the lowest GPX activity and GPX1 mRNA of all tissues examined, but had 15-fold higher PHGPX activity and 45-fold higher PHGPX mRNA levels when compared with liver. There was no significant effect of dietary Se on testis GPX1 and PHGPX mRNA levels. This study demonstrates that these two selenoperoxidases are differentially regulated by dietary Se.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.

Animals↗