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At least 1,333 records · Page 74Linked to original sources
PubMed1981

Thyroid hormone synthesis in thyroglobulin. The mechanism of the coupling reaction.

[U-14C]Tyrosine-labeled noniodinated hog thyroglobulin was iodinated enzymatically and nonenzymatically (iodine, iodide-chloramine-T, pH 7.4, or iodine monochloride, pH 8.1). This led to similar levels of iodine incorporation as well as of thyroid hormone synthesis. Iodine monochloride at pH 5.5 formed "hormonogenic" iodotyrosine residues, but no hormone residues. The latter were formed when the iodinated thyroglobulin was brought to pH 8.5 and then treated with horseradish peroxidase and glucose-glucose oxidase in the absence of iodide and iodine monochloride. Enzymatic hydrolysates contained labeled hormone and pyruvic acid; acid hydrolysates labeled thyronine and acetic acid. (Treatment with acid converts hormone to thyronine and pyruvic to acetic acid.) After borohydride treatment, labeled alanine was present instead of pyruvic or acetic acid. The pyruvic acid/hormone, acetic acid/thyronine, alanine/hormone, and alanine/thyronine molar ratios always were 1, independently of the method of iodination. The "coupling reaction" consists of an oxidation step and nonoxidative coupling and decomposition steps. The oxidation step may be either enzymatic or nonenzymatic. The decomposition step always leads to 1 dehydroalanine residue for each hormone residue synthesized. (Dehydroalanine residues appear in the various hydrolysates as acetic acid, pyruvic acid, and alanine, respectively.) Since proper alignment of 2 iodotyrosine residues is a prerequisite for coupling, a model is proposed according to which oxidation of hormonogenic iodotyrosine residues leads to a charge transfer complex which is the same zwitterion-biradical resonance hybrid no matter whether it resulted from a free radical (enzymatic) or an ionic (nonenzymatic) oxidation.

Animals↗
PubMed1978

Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells.

Procedures for the determination of free and total cholesterol in lipid extracts or sonicates of 10(4) cultured human skin fibroblasts are described. The method for free cholesterol employs cholesterol oxidase to generate H2O2 and peroxidase to catalyze the reaction of H2O2 with p-hydroxyphenylacetic acid to yield a stable fluorescent product. Cholesterol ester hydrolase is included for determination of total cholesterol. When samples of sonified cell suspensions are used directly, the extraction of lipids is avoided, permitting one person to carry out analyses of 30 or more subcultures in one day.

Cells, Cultured↗
PubMed1975

The histochemistry of armadillo skin.

The histochemistry of armadillo skin has been studied. The dendritic cells are extremely large, very sharply outlined by methods for alkaline phosphatase and alpha-naphthyl-acetate esterase, and they are dopa-negative. The mastocytes, however, are dopa-oxidase-positive, probably due to peroxidase rather than tyrosinase activity. The giant cells of the granulomas normally seen in the dermis of the armadillo are strongly beta-glucuronidase-positive. These giant cells are evidently foreign body cells reacting to the crystals always present in the dermis of the armadillo. The centre of these crystals, which are cholesterol and fat-negative, is alkaline phosphatase-positive. Further study of the mastocytes and dendritic cells is necessary to elucidate their nature.

Acetylcholinesterase↗
PubMed1971

Isolation and properties of phagocytic vesicles from polymorphonuclear leukocytes.

A method for the isolation of intact phagocytic vesicles from guinea pig peritoneal-exudate granulocytes and human peripheral-blood leukocytes is presented. After leukocytes ingested the particles of a stable emulsion of paraffin oil, the uningested emulsion was washed away and the cells were homogenized. The homogenate was placed in the middle of a three-step discontinuous sucrose gradient and centrifuged for 1 hr at 100,000 g. The phagocytic vesicles, containing the low density paraffin-oil particles, were simultaneously washed and collected by floatation, while the other organelles, chiefly granules, sedimented through the lower wash layer, and the particle-free supernatant remained in the middle of the gradient. Emulsion particles stained with Oil Red O were employed to assay the rate of phagocytosis and to mark the location of the particles in subcellular fractions. The dye was extracted from washed cells or cell fractions with dioxane and colorimetrically quantified. The purity of phagocytic vesicles obtained by this method was assessed by electron microscopy, chemical analysis, and assay of enzyme composition. Granule-associated enzymes, acid phosphatase, alkaline phosphatase, beta-glucuronidase, and peroxidase were present in the phagocytic vesicles and originated from the granules. Cyanide-resistant NADH (reduced form of diphosphopyridine nucleotide) oxidase was also found. Enzymes associated with the vesicles exhibited latency to Triton X-100. Uptake of particles and the transfer of total protein and phospholipid into phagocytic vesicles occurred simultaneously Accumulation of acid and alkaline phosphatase in the vesicles continued until phagocytosis ceased. Peroxidase, NADH oxidase, and beta-glucuronidase activities in the phagocytic vesicles, on the other hand, were maximal by 30 min and increased little thereafter even when phagocytosis was still going on.

Acid Phosphatase↗
PubMed2004

Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth.

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.

Cell Enlargement↗
PubMed2003

Mechanisms of metalloenzymes studied by quantum chemical methods.

The study of metalloenzymes using quantum chemical methods of high accuracy is a relatively new field. During the past five years a quite good understanding has been reached concerning the methods and models to be used for these systems. For systems containing transition metals hybrid density functional methods have proven both accurate and computationally efficient. A background on these methods and the accuracy achieved in benchmark tests are given first in this review. The rest of the review describes examples of studies on different metalloenzymes. Most of these examples describe mechanisms where dioxygen is either formed, as in photosystem II, or cleaved as in many other enzymes like cytochrome c oxidase, ribonucleotide reductase, methane mono-oxygenase and tyrosinase. In the descriptions below high emphasis is put on the actual determination of the transition states, which are the key points determining the mechanisms.

Amine Oxidase (Copper-Containing)↗
PubMed1974

Peroxidatic oxidation of tyrosine to melanin in supernatant of crude mouse melanoma homogenates.

Peroxide-dependent enzymic oxidation of tyrosine to dopachrome and melanin was demonstrated in cell-free melanoma homogenates. Histochemical methods for distinguishing peroxidase activity from aerobic dopa (3,4-dihydroxyphenylalanine) oxidase activity are not reliable with cell-free preparations. Therefore the presence of peroxidase activity in such preparations precludes assay of cresolase activity of mammalian ;tyrosinase'.

Animals↗
PubMed1989

Oxygen activation and defence against oxygen toxicity in a psychrophilic Bacteroidaceae.

When suddenly exposed to air the growth of the obligate anaerobic bacterium of the bacteroidaceae type, strain B6, continues for a few hours before coming to a complete stop. When air is shut off soon after growth has ceased, the organism is able to reestablish anaerobic conditions due to an ability to reduce O2, and resumes normal growth after another few hours. The O2 reducing ability of the organism is due to the presence in the cells of a particle-bound NADH oxidase, a soluble NADPH oxidase and a soluble pyruvate oxidase. The two pyridine nucleotide oxidases reduce O2 to H2O2, the pyruvate oxidase reduces O2 to H2O. Catalase and peroxidase were not detected in anaerobically grown cells. Kinetic studies with cell-free extracts showed that the pyruvate oxidase had a considerably greater affinity (smaller Km) for O2 and capacity (higher Vmax) for O2 reduction than the two other oxidases. It is postulated that the pyruvate oxidase acts as a scavenger for O2, leading to the non-toxic reduction product H2O, and thus functions as a defense mechanism against oxygen toxicity when the organism is exposed to aerobic condition.

Air Microbiology↗
PubMed1999

Activities of DNA turnover and free radical metabolizing enzymes in cancerous human prostate tissue.

Activities of adenosine deaminase (ADA), 5'nucleotidase (5'NT), xanthine oxidase (XO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) and levels of thiobarbituric acid reagent substances (TBARS) were measured in 10 cancerous and 10 noncancerous human prostate tissues. Decreased activities of DNA turnover enzymes (ADA and 5'NT), increased activities of GSH-Px and CAT, and unchanged activities of SOD and XO were observed in cancerous prostate tissues compared with those of noncancerous ones. TBARS levels were found to be higher in cancerous tissues than noncancerous ones. In correlation analysis, mostly positive correlations were established between enzyme activities of the cancerous tissues, whereas no meaningful correlations were found between enzyme activities of the noncancerous tissues except for a positive correlation between XO and SOD. The results indicate that the activities of DNA turnover enzymes were reduced, which was possibly an attempt to lower the rate of purine catabolism, and the activities of GSH-Px and CAT enzymes were increased, probably in response to increased free radical stress occurring in cancerous prostate tissues. Increased concentrations of TBARS suggested oxidant stress and thus accelerated peroxidative reactions in the cancerous tissues, even though antioxidant defense mechanisms were activated. These findings suggest that enzymatic antioxidant systems of cancerous prostate tissues cannot sufficiently eliminate oxidant factors and prevent cellular peroxidative reactions occurring during the carcinogenic process.

5'-Nucleotidase↗
PubMed1996

Application of xanthine oxidase-catalyzed luminol chemiluminescence in a mouse interleukin-5 immunoassay.

A chemiluminescent substrate reagent for use in a sandwich immunoassay for the model antigen mouse interleukin-5 (IL-5) was developed using xanthine oxidase and luminol. Various parameters involved in this chemiluminescent reaction have been studied, including the substrate hypoxanthine, luminol and the Fe(II)-EDTA complex. Addition of the Fe(II)-EDTA complex enhances the chemiluminescence signal considerably. The xanthine oxidase-catalyzed chemiluminescent immunoassay was compared to horseradish peroxidase-linked immunoassays with luminol as chemiluminescent, and tetramethyl benzidine as colorimetric substrate. The detection limit of the xanthine oxidase-luminol assay was found to be about 0.6 pg/ml IL-5, whereas the peroxidase-catalyzed immunoassays have detection limits of about 1.3 (HRP-TMB) and 2.9 pg/ml (HRP-luminol) IL-5.

Animals↗
PubMed1987

An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique.

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).

Alkaline Phosphatase↗
PubMed1986

Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium.

Ligninase-I (Mr 42,000-43,000; carbohydrate, 21%) and peroxidase-M2 (Mr 45,000-47,000; carbohydrate, 17%), two representative, hydrogen peroxide-dependent extracellular enzymes produced by ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium BKM-F-1767, were purified and their properties compared. Spectroscopic studies showed that both native enzymes are heme proteins containing protoporphyrin IX. EPR spectroscopy indicated that iron ions are coordinated with the enzymes' prosthetic groups as high-spin ferriheme complexes. We confirmed reports of others that the ligninase-hydrogen peroxide complex (activated enzyme) reverts to its native state on addition of dithionite or one of the enzyme's substrates (e.g., veratryl alcohol); however, we found that the peroxidase-M2-hydrogen peroxide complex required Mn2+ ions to accomplish a similar cycle. The peroxidase oxidized Mn2+ to a higher oxidation state, and the oxidized Mn acted as a diffusible catalyst able to oxidize numerous organic substrates. Unlike ligninase-I which is found free extracellularly, peroxidase-M2 appears to be associated closely with the fungal mycelium. In its peroxidatic reactions, ligninase-I oxidizes a variety of nonphenolic and phenolic lignin model compounds. In the presence of Mn2+, peroxidase-M2 oxidizes numerous phenolic compounds, especially syringyl (3,5-dimethoxy-4-hydroxyphenyl) and vinyl side-chain substituted substrates. Also, the peroxidase-Mn2+ system (without hydrogen peroxide) expresses oxidase activity against NADPH, GSH, dithiothreitol, and dihydroxymaleic acid, forming hydrogen peroxide at the expense of oxygen. Both enzymes were believed to play roles in lignin degradation, and these are discussed.

Amino Acids↗
PubMed1998

Separation-free electrochemical immunosensor for rapid determination of atrazine.

A separation-free electrochemical immunoassay method for the detection of the pesticide atrazine is described. The method developed is a competitive ELISA incorporating disposable screen printed horseradish peroxidase modified electrodes as the detector element in conjunction with single-use atrazine immuno-membranes. Screen printed carbon electrodes were prepared using carbon ink incorporating horseradish peroxidase. A monoclonal antibody for atrazine was immobilised onto Biodyne C membranes which were, in turn, placed over the electrode surface. The assay was based on competition for available binding sites between free atrazine and an atrazine-glucose oxidase conjugate prepared 'in-house'. In the presence of glucose, H2O2 formed by the conjugate was reduced by enzyme-channelling via the HRP electrode. The HRP was in turn re-reduced by a direct electron transfer mechanism at a potential of +50 mV Vs Ag/AgCl. Any H2O2 formed in the bulk solution by unbound atrazine-GOD conjugate was scavenged by excess catalase thus removing the requirement for a washing step. The performance of the method was compared with a commercial immunoassay kit for atrazine.

Antibodies, Monoclonal↗
PubMed1975

Studies on the decomposition of lignosulfonates by the fungi Pleurotus ostreatus and Trametes pubescens.

The fungi Pleurotus ostreatus and Trametes pubescens were grown in a mineral medium containing 1% of glucose and 0.9% of lignosulfonates introduced into the culture medium in the form of yeast waste liquor. Chromatography of extracts of the medium and determinations of sulphur and lignosulfonates have revealed that the fungi studied utilized the constituents of the yeast waste liquor (lignosulfonates) as carbon source. This was manifested in an increase of dry mass of the mycelium and protein as compared with the control. The constituents of the yeast waste liquor were also found to have a stimulating effect on the formation of both exo-and endoenzymes, laccase and peroxidase. This may indicate that these oxidases take part in the decomposition of lignosulfonates.

Agaricales↗