Cell Wall Free Space of Cucumis Hypocotyls Contains NAD and a Blue Light-Regulated Peroxidase Activity

Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6-phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related.     

Proof of plasmatic imbibition in rat musculocutaneous grafts: enzymatic proof using peroxidase.

At present, the concept of plasmatic imbibition is generally accepted. That is, observation of weight change or pigmentation methods of the grafts were established to support it. In the present study, we investigated plasmatic imbibition in rat musculocutaneous grafts histologically using peroxidase, which is one of the reductases. Tissue pigmentation by peroxidase became an insoluble sediment that indicated the sites of peroxidase activity. The entire contact surface of the graft with the recipient bed was stained brown within a few minutes after the operation. At 30 minutes, the panniculus carnosus and dermis were stained dark brown diffusely, extending toward the epidermis. This condition continued until the sixth day at least. As a result, peroxidase that was dissolved with the exudate between the graft and recipient bed was imbibed into the musculocutaneous graft.     

Application of the Electron Microscope to the Cytochemical Peroxidase Reaction in Salamander Leukocytes

The present study has dealt with the localization by electron microscopy of the products of peroxidase reaction in neutrophil leukocytes in the subcapsular region of the livers of Triturus viridescens. Small pieces of liver tissue were fixed for 1 hour in buffered osmium tetroxide solution. After fixation they were divided into five groups: (a) Not treated with any reagent (control); (b) Treated for 4 minutes with the peroxidase reagent containing 0.3 per cent benzidine and 0.014 per cent (0.004 molar) hydrogen peroxide in 50 per cent alcohol; (c) Treated for 4 minutes with 0.3 per cent benzidine solution in 50 per cent alcohol alone (control); (d) Treated for 4 minutes with 0.014 per cent (0.004 molar) hydrogen peroxide in 50 per cent alcohol alone (control); (e) Treated for 5 minutes with pure methanol, washed in water, and treated for 4 minutes with the peroxidase reagent (inhibition test). Each group was then dehydrated and embedded in either methacrylate or epoxy resin. In electron micrographs, the reaction products of peroxidase activity were evidenced in the form of dense materials localized in the specific granules in the cytoplasm of the neutrophil leukocytes. Neither mitochondria nor any other particles showed increases in density. The specific granules showed no change of density in the control and inhibition tests. Paraffin-embedded tissues of the above mentioned five groups, when examined with the light microscope, revealed that the brown granules denoting a positive reaction appeared only in leukocytes of the tissue treated with the peroxidase reagent. Although much further work is necessary before definitive and constant results are to be expected, the possibility that the electron microscope may be applicable to peroxidase cytochemistry in leukocytes has been suggested by the present study.     

Absorption of peroxidase by osteoclasts as studied by electron microscope histochemistry

Summary The ability of osteoclasts to take up protein by endocytosis was examined using peroxidase as a tracer. 5 minutes after intravenous injection the tracer was located around the osteoclast and in the space between its ruffled border and the bone. Inside the cell peroxidase was located in some cytoplasmic vacuoles behind the ruffled border and along the cell membrane. 40 minutes after injection there was a large increase in the number of membrane limited cytoplasmic structures containing reaction product, these being distributed in general throughout the cell but with a high concentration behind the ruffled border. These structures which were filled throughout with peroxidase represented either vacuoles or bodies.The study demonstrates, first that the osteoclast is able to absorb peroxidase, second that a transport of material occurs from the periphery towards the central part of the cell. From the extensive endocytosis along the ruffled border, where the bone resorption takes place, it is suggested that also organic components of the bone may be taken up by the osteoclast under bone resorption in a manner similar to that for peroxidase.This research was supported by the Danish Research Council. Grant. no. 512-819. I wish to thank Mrs. Ruth Nielsen for skilful technical assistance during this work.     

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation

Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249-264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.     

An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit

A conventional colorimetric peroxidase end-point (ortho-phenylenediamine substrate), used in an enzyme immunoassay for carcinoembryonic antigen, employing plastic beads as solid support, has been replaced by a much faster (30 seconds versus 30 minutes) enhanced chemiluminescent assay for the peroxidase label. Para-iodophenol was used to enhance the light emission from the peroxidase catalysed chemiluminescent reaction between luminol and hydrogen peroxide. Values for precision and carcinoembryonic antigen concentration obtained with the chemiluminescent and colorimetric versions of the immunoassay on 62 serum specimens were in good agreement.     

Immunological detection of adulteration of ground meats by meats of other origins

Specific and sensitive assays for immunoglobulin G in pork, chicken, and beef were developed using polyester cloths coated with respective anti-immunoglobulin G antibodies. The captured immunoglobulin G proteins were detected by their reactions with the respective IgG antibody horseradish peroxidase conjugates and a chromogenic peroxidase substrate. This sensitive assay takes only 30 minutes and can detect the adulteration of ground meats.     

Anions activate the oxidation of indoleacetic Acid by peroxidases from tomato and other sources

Anionic peroxidase from tomato (Lycopersicon esculentum) fruit oxidized indoleacetic acid (IAA) slowly in the presence of Mn²⁺ and dichlorophenol in acetate buffers. The addition of certain anions to the reaction mixture increased the rate of oxidation. Phosphate was one of the effective anions and exerted maximal activation at 0.1 molar. The most effective activator of tomato peroxidase was nitrilotriacetate (NTA) at an optimum concentration of 60 micromolar. Only 0.17 nanomolar peroxidase was needed to oxidize 0.1 micromole IAA/5 minutes in the presence of NTA compared to 650 nanomolar peroxidase for the same rate in the absence of NTA. Other effective anions were oxalate, pyrophosphate, malate, and citrate. Each activator exhibited an optimum concentration and higher concentrations were inhibitory. Anionic peroxidase from horseradish was activated by the same anions. A cationic peroxidase from horseradish and lactoperoxidase oxidized IAA in acetate buffer although anions activated these enzymes severalfold. Microperoxidase and other hematoporphrins also catalyzed IAA oxidation in the presence of anions. It is proposed that IAA oxidation by peroxidase may be important when vacuolar contents mix with peroxidase as during plant injury.     

Differential Activation of Expression of a Suberization-Associated Anionic Peroxidase Gene in Near-Isogenic Resistant and Susceptible Tomato Lines by Elicitors of Verticillium albo-atratrum

We tested whether the expression of the suberization-associated anionic peroxidase gene is involved in the timely appearance of the vascular suberized coating involved in the resistance of a tomato line to Verticillium albo-atrum. The mRNA for this peroxidase appeared at a higher level one day earlier in wound-healing fruits of the resistant tomato line than in a near-isogenic susceptible line. Cell cultures from the resistant line, when treated with low levels (nanograms per milliliter) of fungal elicitor, generated the peroxidase mRNA and this apparent activation of the peroxidase gene expression could be detected in minutes, whereas the cells from the susceptible line hardly responded.     

Chemical stabilization of recombinant horseradish peroxidase

Summary Unglycosylated recombinant horseradish peroxidase (HRP C^*) had a half life of 21 minutes at 65°C compared with only 5 minutes for the plant enzyme (HRP). The half life of HRP C^* at 65°C increased by 5-fold following modification with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Tolerance to 60% 1,4-dioxan also increased whilst tolerance to 30% dimethylformamide was unchanged.     

Über den Peroxydasetransport im Darmepithel der Ratte

Zusammenfassung Bei Ratten tritt 3 min nach intravenöser Injektion von Peroxydase elektronenmikroskopisch ein entsprechendes Reaktionsprodukt im Kapillarlumen der Lamina propria des Dünndarms und an der Basalmembrangrenze der Saumepithelzellen auf. 5 min nach der Injektion finden sich im basalen Abschnitt des Darmepithels pinozytotische Bläschen mit dem Peroxydase-Reaktionsprodukt. — 10–30 min nach der Injektion erreichen die Partikel die apikalen Teile der Zelle. Sie dringen in den interzellulären Spalten bis zu den Haftplatten vor, erreichen jedoch nie das Darmlumen. Im Dünndarm existiert vermutlich auch ein der Resorption entgegengesetzter Saftstrom, der durch Peroxydase markiert werden kann.

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The transport of horseradish peroxidase in the epithelium of the small intestine

Summary In rats, 3 minutes after intravenous injection of peroxidase the reaction product can be observed electronmicroscopically in the lumina of the capillaries of the small intestine as well as at the border of the basement membrane of the epithelial border cells. Pinocytotic vesicles containing peroxidase particles occur in the basal portion of the epithelium of the small intestine 5 minutes after injection. 10–30 minutes later, the peroxidase reaches the apical region of the cell. The particles infiltrate into the intercellular spaces as far as the tight junctions but never reach the intestinal lumen. In the small intestine there probably exists a flow of fluid in opposite direction to the resorption, which can be marked by peroxidase.

     

The distribution of peroxidase in phagocytizing human blood platelets, electron microscopy findings

On the basis of a previously elaborated method for testing the phagocytic ability of human blood platelets we have undertaken studies for testing the distribution of peroxidase in phagocytizing platelets. Our findings confirmed the distribution of peroxidase in the dense tubular system and in granules in regular blood platelets. In the course of phagocytosis we noted the presence of peroxidase less evidently in both subcellular structures during the first of 5 minutes. In the next steps we observed the localization of this enzyme mainly near the phagolysosome. The change of this localization and the role of this enzyme in unspecific immunity against bacteria and neoplasia is briefly discussed.     

A method of fast separation of lignin peroxidases using convective interaction media disks.

The HPLC separation of lignin peroxidase isoenzymes using Convective Interaction Media disks containing quaternary amine and diethylaminoethyl ion-exchange active groups is proposed. In contrast to standard HPLC procedures the separation can be performed within a few minutes without considerably affecting the separation resolution. The method is reproducible and gives a linear response of integrated peak area to protein concentration for all measured isoenzymes. The separation resolution is retained unchanged by applying crude culture filtrate instead of a sample previously frozen and dialyzed. The optimized method might therefore be used for on-line monitoring of lignin peroxidase isoenzyme composition during fermentation. On the other hand, the proposed method is comparable in time to the original method of lignin peroxidase activity measurement (proposed by Tien and Kirk), providing additionally the isoenzyme composition.     

Ultrastructural investigation of the meningeal compartment of the blood-cerebrospinal fluid-barrier in rats and cats. A horseradish peroxidase study

The permeability of the meningeal blood vessels and cellular layers to horseradish peroxidase was studied 5, 10 and 15 minutes following intravasal or intraarachnoidal introduction of the marker. When applied intravasally, the horseradish peroxidase-containing solution easily passed through the walls of all meningeal vessels (dural, pial and the ones traversing the arachnoid space). The cells of the inner dural layer and dural neurotheliun delay the penetration of horseradish peroxidase into the cerebrospinal fluid-filled arachnoid space by 10 min--rats and 15 min--cats. The perivascular leptomeningeal cells and their processes restrict the passage of the marker into the arachnoid space in a similar way. These barrier functions of the leptomeningeal cells and the cells that comprise the interface zone between dura mater and the arachnoid are confirmed by experiments where the marker was injected into the arachnoid space.     

NADH peroxidase activity of rubrerythrin

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.     

Inactivation of trypsin and chymotrypsin with a photosensitive probe.

The photosensitive inactivation of trypsin and chymotrypsin by 4-fluoro-3-nitrophenyl azide (FNPA) is described. A dark inhibition was observed at elevated probe concentrations, and was reversible. The enzymes were stable to photolysis in the absence of probe. Photolytic inactivation of trypsin and chymotrypsin with FNPA was found to be irreversible, and occurs in minutes at concentrations of FNPA where dark inhibition is negligible. The photoprobe was equally effective at pH 3 or pH 8. Nonspecific inactivation appears to be low, as evidenced by the stability of glucose oxidase and peroxidase to photolysis with FNPA.     

Incorporation of a circulating protein into alpha granules of megakaryocytes

In order to determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for uptake by cytochemistry and electron microscopy. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare bone marrow cells. In megakaryocytes, more than 50% of alpha granules contained HRP between 75 minutes and 7 hours after injection. At 24 hours, 25% of the megakaryocyte granules were peroxidase positive; less were so by 48 hours and none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. A precipitous drop in circulating platelet numbers was observed 45 minutes after injection. At this time, circulating platelets showed the tracer only on the platelet plasma membrane, and none in platelet granules. Platelet numbers increased to 35% by 7 hours and only the platelet granules contained HRP. These platelets secreted the HRP stored in granules in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. Our studies are the first to demonstrate an endocytic pathway by which megakaryocytes can incorporate a circulating protein into alpha granules. An important physiologic implication of this endocytic pathway is the possible origin of certain alpha granule proteins from plasma.     

Techniques for histological reconstruction of brain stem recording sites.

The masseter muscle of Sprague-Dawley rats was injected with 10 mg of horseradish peroxidase. The electrical activity of the trigeminal motor nucleus was investigated 24-36 hours later using micropipettes filled with a 2.0 M NaCl-7% fast green FCF solution. A parallel series of penetrations at 200 micron intervals was made through the motor complex at known depths. The grid formed from these penetrations was reconstructed using the following techniques. Marks were made at known depths in one or more of the tracts by iontophoresing fast green from the microelectrode tip with a hyperpolarizing current of 10 muA for 10 minutes. To assure proper alignment of the tissue containing the green marks, two agar-India ink plugs were placed caudal and parallel to the recording sites. The frozen tissue was sectioned on a microtome, first through the agar plugs and then through the tissue containing the green marks. The tissue could be incubated for the peroxidase reaction product or stained for Nissl substance. These combined procedures offer a means to correlate the structure and function of brain stem nuclear groups.     

Studies on Auxin Protectors: XI. Inhibition of Peroxidase-Catalyzed Oxidation of Glutathione by Auxin Protectors and o-Dihydroxyphenols

Commercial horseradish peroxidase, when supplemented with dichlorophenol and either manganese or hydrogen peroxide, will rapidly oxidize glutathione. This peroxidase-catalyzed oxidation of glutathione is completely inhibited by the presence of auxin protectors. Three auxin protectors and three o-dihydroxyphenols were tested; all inhibited the oxidation. Glutathione oxidation by horseradish peroxidase in the presence of dichlorophenol and Mn is also completely inhibited by catalase, implying that the presence of Mn allows the horseradish peroxidase to reduce oxygen to H₂O₂, then to use the H₂O₂ as an electron acceptor in the oxidation of glutathione. Catalase, added 2 minutes after the glutathione oxidation had begun, completely inhibited further oxidation but did not restore any gluthathione oxidation intermediates. In contrast, the addition of auxin protectors, or o-dihydroxyphenols, not only inhibited further oxidation of gluthathione by horseradish peroxidase (+ dichlorophenol + Mn), but also caused a reappearance of glutathione as if these antioxidants reduced a glutathione oxidation intermediate. However, when gluthathione was oxidized by horseradish peroxidase in the presence of dichlorophenol and H₂O₂ (rather than Mn), then the inhibition of further oxidation by auxin protectors or o-dihydroxyphenols was preceded by a brief period of greatly accelerated oxidation. The data provide further evidence that auxin protectors are cellular redox regulators. It is proposed that the monophenol-diphenol-peroxidase system is intimately associated with the metabolic switches that determine whether a cell divides or differentiates.     

酶标免疫分析法鉴定蛇毒

蛇伤的治疗与蛇伤种类的快速诊断密切相关。我们用亲和层析法纯化了三种蛇毒的抗体Fab,它们之间无免疫交叉反应。然后用高碘酸钠法将辣根过氧化物酶标记到抗体Fab上。我们建立的三夹心式酶标免疫测定蛇毒的方法,可在90分钟内检测到5毫微克以下的蛇毒,人体血清对该测定无干扰现象,临床检测效果良好。