Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.3 (ascorbate oxidase)
778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.
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PMID:Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties. 22 38

Ascorbate (0-500 microM) stimulates synthesis and secretion of collagen by cartilage explants in a dose-dependent fashion as estimated by [3H]proline incorporation. Concurrent elaboration of inorganic pyrophosphate (PPi) parallels [3H]proline incorporation (less than 0.001, Wilcoxon rank sum). The effect on proline and PPi is abolished by ascorbate oxidase. Another reducing agent, Vitamin E, did not promote PPi accumulation about cartilage. Inhibitors of collagen synthesis, including monensin, tumor necrosis factor alpha, and 2',2'dipyridyl also inhibited PPi elaboration. Cycloheximide (1 micrograms/ml) inhibited the ascorbate stimulated PPi elaboration 54% but did not attenuate the secretion of PPi by unstimulated cartilage. Cosecretion of collagen and PPi by chondrocytes may explain these results. Moreover, at least two pathways exist for PPi elaboration, a cycloheximide sensitive path and another independent of new protein synthesis.
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PMID:Stimulation of cartilage inorganic pyrophosphate elaboration by ascorbate. 165 74

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 microM) or riboflavin (500 microM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.
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PMID:Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium. 222 47

Ascorbate oxidase was immobilised on cyanogen bromide activated-Sepharose 4B and incorporated in a flow-injection system with amperometric detection at a glassy carbon electrode at +0.6 V. On passage through the immobilised ascorbate oxidase a fraction of the L-ascorbic acid was converted into dehydroascorbic acid and the decrease in signal was measured. This could be directly related to the amount of L-ascorbic acid present. The calibration graph was linear over the range 0-400 ng ml(-1) with a correlation coefficient of 0.9994. The detection limit (2 sigma) in phosphate buffer (0.08 M, pH 5.5) was 4.0 ng ml(-1). The relative standard deviation for a 200 ng ml(-1) standard was 1.0% (n = 10) and the sampling throughput was 30 samples h(-1). The method was used for the simple and rapid determination of L-ascorbic acid in fruit and vegetable juice.
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PMID:Determination of L-ascorbic acid in fruit and vegetable juices by flow injection with immobilised ascorbate oxidase. 227 Aug 72

A preliminary study (J.M. Mata, R. Assad, and B. Peterkofsky (1981) Arch. Biochem. Biophys. 206, 93-104) suggested that chick embryo limb bone microsomes took up and concentrated [14C]ascorbate in the presence of cofactors for prolyl hydroxylase. In the present study, we found that the apparent Km for ascorbate in the hydroxylation of intracisternal unhydroxylated procollagen by endogenous prolyl hydroxylase was approximately an order of magnitude less than the value obtained when enzyme solubilized from microsomes was used with an exogenous substrate. These results are compatible with a concentrative uptake of ascorbate into microsomes. The uptake of [14C]ascorbate into microsomes was confirmed and it required only iron, in either the ferrous or ferric form, and was time and temperature dependent, proportional to microsome concentration, and substrate saturable at 2-3 mM ascorbate. Iron-dependent ascorbate uptake also was observed with L-929 cell microsomes. [14C]Ascorbate seemed to be taken up without prior oxidation, since only unlabeled ascorbate, and not dehydroascorbate, competed for uptake into limb bone microsomes. A functional requirement for Fe2+ in ascorbate transport was demonstrated using the intracisternal proline hydroxylating system. L-929 cell microsomes were preincubated with ascorbate with or without the metal and then external ascorbate was oxidized to inactive dehydroascorbate using ascorbic acid oxidase, which cannot penetrate the microsomal membrane. Samples which did not receive iron during the preincubation received it, along with other requirements for prolyl hydroxylase, in a final incubation to measure hydroxylation. Significant hydroxylation was obtained only in samples incubated with iron prior to oxidase treatment, consistent with the conclusion that an iron-dependent process was required to translocate ascorbate and protect it from the oxidase.
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PMID:Iron-dependent uptake of ascorbate into isolated microsomes. 303 60

Rat Schwann cells cultured with dorsal root ganglion neurons in a serum-free defined medium fail to ensheathe or myelinate axons or assemble basal laminae. Replacement of defined medium with medium that contains human placental serum (HPS) and chick embryo extract (EE) results in both basal lamina and myelin formation. In the present study, the individual effects of HPS and EE on basal lamina assembly and on myelin formation by Schwann cells cultured with neurons have been examined. Some batches of HPS were unable to promote myelin formation in the absence of EE, as assessed by quantitative evaluation of cultures stained with Sudan black; such HPS also failed to promote basal lamina assembly, as assessed by immunofluorescence using antibodies against laminin, type IV collagen, and heparan sulfate proteoglycan. The addition of EE or L-ascorbic acid with such HPS led to the formation of large quantities of myelin and to the assembly of basal laminae. Pretreatment of EE with ascorbic acid oxidase abolished the EE activity, whereas trypsin did not. Other batches of HPS were found to promote both basal lamina and myelin formation in the absence of either EE or ascorbic acid. Ascorbic acid oxidase treatment or dialysis of these batches of HPS abolished their ability to promote Schwann cell differentiation, whereas the subsequent addition of ascorbic acid restored that ability. Ascorbic acid in the absence of serum was relatively ineffective in promoting either basal lamina or myelin formation. Fetal bovine serum was as effective as HPS in allowing ascorbic acid (and several analogs but not other reducing agents) to manifest its ability to promote Schwann cell differentiation. We suggest that ascorbic acid promotes Schwann cell myelin formation by enabling the Schwann cell to assemble a basal lamina, which is required for complete differentiation.
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PMID:Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. 362 5

Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide. The properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxidase. The immobilized enzyme was utilized in a flow-through system equipped with a polarographic detector which monitors the oxygen depletion due to the reaction ascorbic acid + 1/2 O2----dehydroascorbic acid + H2O. This method, the response of which is linear between 3 X 10(-7) and 5 X 10(-4) M ascorbate, was utilized to measure the ascorbic acid in biological samples such as human plasma and fruit juices at a rate of about 60 determinations every hour with a standard deviation lower than 5%.
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PMID:Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase. 407 8

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.
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PMID:A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa. 664 10

High speed cyclic voltammetry was used in combination with pressure ejection of ascorbate oxidase for the determination of extracellular ascorbic acid within the brain of the anaesthetized rat. Large variations in absolute levels of ascorbate were found between animals although distribution patterns showed a good degree of reproducibility. Ascorbate levels in the white matter of the corpus callosum were found to be higher than in adjacent areas of grey matter (striatum and cortex).
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PMID:Regional differences in extracellular ascorbic acid levels in the rat brain determined by high speed cyclic voltammetry. 673 51

We describe a specific enzymatic spectrophotometric method for ascorbic acid in serum or plasma. Samples are analyzed indirectly by measuring the absorbance at 593 nm of a reaction product, a complex of ferrous ion and 2,4,6-tris(2-pyridyl)-s-triazine (Fe2+-TPTZ). This product is formed by reduction of the corresponding ferric ion complex (Fe3+-TPTZ), which is nonspecifically reduced by various biological reducing agents under acidic conditions. Ascorbic acid is specifically quantified by pretreating one of a pair of replicate samples with ascorbate oxidase (EC 1.10.3.3), to oxidize the ascorbic acid, then reacting both samples with Fe3+-TPTZ and measuring the difference between the absorbances at 593 nm of the treated and untreated samples. This difference is linearly related to ascorbic acid concentrations from 10 to 100 mg/L. Ten repeat determinations of a serum pool with added ascorbic acid yielded a CV of 2.8% and a mean of 47.2 mg/L. The correlation (r) between the proposed method and the dinitrophenylhydrazine method was 0.93 for 32 samples analyzed by both methods. The present method is specific for ascorbic acid and requires no deproteinization.
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PMID:Specific spectrophotometry of ascorbic acid in serum or plasma by use of ascorbate oxidase. 712 67


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