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PubMed1995

Direct observation of the action of cholesterol oxidase in monolayers.

The oxidation of monolayer cholesterol by cholesterol oxidase has been visualized using monolayer fluorescence microscopy. A direct microscopic visualization was possible because the lateral distribution of a lipid fluorophore, tetramethylrhodamine (TRITC)-labeled phosphatidylethanolamine, was very different in a cholesterol containing monolayer as compared with a cholestenone monolayer. The lipid fluorophore was effectively excluded from the condensed cholesterol phase, but was readily miscible in the cholestenone phase. One could therefore observe the appearance of fluorophore rich cholestenone-domains in the cholesterol monolayer as a result of the cholesterol oxidase catalyzed oxidation reaction. The oxidation experiments were performed at 22 degrees C with a monolayer surface pressure of 5 mN/m (on 50 mM Tris-HCl buffer, containing 140 mM NaCl, pH 7.4). When 40 mU/ml of cholesterol oxidase was injected beneath the monolayer under observation, it appeared that the enzyme penetrated the cholesterol monolayer at random sites and initiated the oxidation reaction. Once the oxidation reaction had commenced, it progressed rapidly and converted the condensed (cholesterol-rich) phase into an expanded (cholestenone-rich) phase. When the oxidation of cholesterol in mixed cholesterol/dimyristoylphosphatidyl-choline monolayers was visualized, it was observed that the enzyme-catalyzed oxidation started from the expanded phases (domains with higher compressibility) and the reaction eventually led to the dissipation of the boundary line between expanded and condensed phases. With time all condensed phases were dissolved and the monolayer became uniformly fluorescent. The association of TRITC-labeled cholesterol oxidase with a non-fluorescent mixed cholesterol/dimyristoylphosphatidylcholine monolayer led to the penetration (or association) of the fluorescent cholesterol oxidase into expanded phases of the mixed monolayers. The monolayer lateral domain morphology was similar whether the fluorescent probe was TRITC-PE or TRITC-labeled enzyme. It is concluded that cholesterol oxidase associated with (or penetrated to some extent into) the expanded phases of a monolayer, and carried out its oxidation reaction in the expanded phase or at the interface between expanded and condensed phases.

Cholestenones↗

PubMed1986

Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate.

Evidences have been provided by many laboratories that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) is strictly linked to a transduction pathway that involves the stimulation, via GTP binding protein, of the phosphoinositide turnover and the increase in [Ca2+]i. The results presented in this paper demonstrate that FMLP can activate the NADPH oxidase by triggering a transduction pathway completely independent of phosphoinositide turnover and Ca2+ changes. In fact: i) Ca2+-depleted neutrophils do not respond to FMLP with the activation of phosphoinositide hydrolysis and NADPH oxidase. Both the responses are restored by the addition of exogenous Ca2+. ii) In Ca2+-depleted neutrophils phorbol-myristate-acetate (PMA) activates the NADPH oxidase. iii) The pretreatment of Ca2+-depleted neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase but not of the turnover of phosphoinositides by FMLP. This priming effect of PMA and the role of this phosphoinositide and Ca2+-independent pathway for the stimulation of the NADPH oxidase by receptors mediated stimuli are discussed.

Calcium↗

PubMed1988

Effect of antibiotics on the binding of pargyline to monoamine oxidase in cultured hepatocytes.

The laser dye rhodamine 123 has been used to establish that the binding of [3H]pargyline to monoamine oxidase is a more sensitive indicator of mitochondrial perturbation than measurements of protein synthesis, secretion, or degradation. The amount of monoamine oxidase labelled depends on the antibiotic used. The labelling was considerably lower in the presence of gentamycin than in the presence of either chloramphenicol or of penicillin and streptomycin. The accumulation of gentamycin within the cells was the cause of the reduced labelling of monoamine oxidase which was not accompanied by an alteration in the metabolism of pargyline. The gentamycin effect can be prevented by incubating the cells in medium supplemented with methylamine prior to adding the monoamine oxidase inhibitor. Long term culture of cells with gentamycin can result in the cell culture medium becoming dark brown. Under these conditions the monoamine oxidase labelling is increased due to an inhibition of pargyline metabolism. The results indicate that the choice of antibiotic is important in patients being treated with both antibiotics and monoamine oxidase inhibitors.

Anti-Bacterial Agents↗

PubMed1991

Methylene blue as an inhibitor of superoxide generation by xanthine oxidase. A potential new drug for the attenuation of ischemia/reperfusion injury.

Tissue oxidases, especially xanthine oxidase, have been proposed as primary sources of toxic oxygen radicals in many experimental models of disease states. Among these, ischemia-reperfusion injury may be of the greatest clinical interest. In this paper we propose the use of methylene blue as a means of suppressing the production of superoxide radicals O2- by acting as an alternative electron acceptor for xanthine oxidase. Previous work has indicated that methylene blue accepts electrons from xanthine oxidase at the iron-sulfur center. Initial experiments in our laboratory demonstrated that (1) pairs of electrons from each enzymatic oxidation are transferred to methylene blue, (2) the reduction of methylene blue can be achieved by model iron-sulfur centers, similar to the iron-sulfur center of xanthine oxidase, (3) reduced methylene blue auto-oxidizes to produce H2O2 directly, rather than O2-, and (4) methylene blue is effective at non-toxic levels (2-5 mg/kg) in preventing free radical damage to liver and kidney tissues in an in vitro model of ischemia and reoxygenation. Accordingly, we propose that methylene blue may represent a new class of antioxidant drugs that competitively inhibit reduction of molecular oxygen to superoxide by acting as alternative electron acceptors for tissue oxidases. We have termed these agents "parasitic" electron acceptors.

Animals↗

PubMed1992

Effects of anti-allergic drugs on human neutrophil superoxide-generating NADPH oxidase.

The effects of anti-allergic drugs with or without H1-receptor antagonism on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Three anti-allergic drugs with H1-receptor antagonism, azelastine, ketotifen and oxatomide, were found to inhibit the superoxide generation of human neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of these drugs required for 50% inhibition of the oxidase (IC50) were: azelastine--0.7 microM in the whole-cell system and 0.5 microM in the cell-free system; ketotifen--60 microM in the whole-cell system and 6.8 microM in the cell-free system; and oxatomide--25 microM in the whole-cell system and 9.7 microM in the cell-free system. In addition, in the cell-free system, these drugs did not change the Km values for the NADPH of the oxidase. However, these drugs did not inhibit the superoxide generation of NADPH oxidase after its activation in whole-cell and cell-free systems, suggesting that these drugs do not have superoxide-scavenger actions. Concentrations of less than 200 microM of anti-allergic drugs without H1-receptor antagonism, tranilast, repirinast and ibudilast, did not inhibit neutrophil NADPH oxidase in whole-cell and cell-free systems. The IC50 of hydrocortisone in the cell-free system was 60 microM. These results suggest that anti-allergic drugs with H1-receptor antagonism inhibit activation of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in cell-free systems and that they have much stronger anti-inflammatory action than hydrocortisone.

Cell-Free System↗

PubMed1994

Dopamine metabolism in the guinea pig striatum: role of monoamine oxidase A and B.

These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig affects the actions of (-)-deprenyl on dopamine metabolism in whole tissue or in extracellular fluid. The administration of (-)-deprenyl in doses that do not affect monoamine oxidase A activity (1-4 mg kg-1, 2 h) increases striatal 2-phenylethylamine and dopamine concentrations and reduces 3,4-dihydroxyphenylacetic acid. No effects were observed on striatal homovanillic acid, 5-HT and 5-hydroxyindole acetic acid. Inhibition of monoamine oxidase A with clorgyline with doses up to 8 mg kg-1 (2 h) does not affect striatal 2-phenylethylamine but increases dopamine and 5-HT concentrations and reduces 3,4-dihydroxyphenyl-acetic acid and 5-hydroxyindole acetic acid. (-)-Deprenyl (2-4 mg kg-1) did not change the extracellular concentrations of dopamine but the higher dose produced a limited reduction in extracellular 3,4-dihydroxyphenylacetic acid. Inhibition of monoamine oxidase A and monoamine oxidase B with pargyline (75 mg kg-1, 2 h) significantly increased the levels of extracellular dopamine and reduced those of their acid metabolites. These results show that in the guinea pig striatum inhibition of monoamine oxidase B by (-)-deprenyl impairs the metabolism of dopamine in the whole tissue but does not produce a marked increase in extracellular dopamine.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1987

Oxidation of N-alkyl and C-alkylputrescines by diamine oxidases.

N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescine were assayed as substrates of diamine oxidase from pea seedling and pig kidney. With the exception of N-methylputrescine they were found to be oxidized to the corresponding aminoaldehydes. 1-Methyl-, 2-methyl-, 1-ethyl- and 1-propylputrescine were oxidized by the oxidases at lower rates than the N-alkylderivatives. 1,3-Dimethylputrescine had negligible oxidation rates while 1,4-dimethylputrescine (2,5-diaminohexane) was not a substrate. The oxidation of putrescine by the kidney oxidase was inhibited by 1,4-dimethylputrescine, while the pea oxidase was strongly inhibited by the former as well as by 2-methylputrescine and 1,3-dimethylputrescine. Serum amine oxidase did not oxidize the substituted putrescines although several of the latter inhibited spermidine oxidation by this oxidase.

Alkylation↗

PubMed1990

Polarized secretion of diamine oxidase by intestinal epithelial cells and its stimulation by heparin.

The Caco-2 cells have been used as a model system to study the pathways of diamine oxidase secretion by the intestinal epithelium. When grown in Transwell filter chamber devices, the polarized cell monolayers released the enzyme preferentially into the basal chamber. Heparin (1-10 USP U/mL) rapidly induced a marked stimulation of enzyme secretion only when in contact with the basolateral cell membrane, where high affinity binding sites for [3H]heparin were also exclusively located. Among the other glycosaminoglycans tested, only heparan sulfate (150 mg/mL) was able to induce enzyme release; chondroitin sulfate (150 mg/mL) and dermatan sulfate (150 mg/mL) were without effect. Four monoclonal antibodies specific for human diamine oxidase were produced and found to immunoprecipitate a single protein with a molecular weight of 95,000 (under reducing conditions) from the culture medium of Caco-2 cells. Immunofluorescence staining of cryostat sections of human small intestine with these four antibodies localized diamine oxidase at the lateral and basal sides of the villus cells. Staining was markedly reduced in specimens obtained from patients who received doses of heparin in vivo. This study concludes that release of diamine oxidase by intestinal cells occurs specifically at the basolateral aspect of the cells, most likely through the constitutive secretory pathway. Heparin may induce its marked stimulation of enzyme release by complexing with diamine oxidase bound to the cell surface or through interaction with specific binding sites also located in the basolateral membrane. In the intestinal mucosa in vivo, the basal aspect of the villus cells represents the main site of diamine oxidase storage in the presence of normal circulating levels of heparin.

Amine Oxidase (Copper-Containing)↗

PubMed1990

Postheparin plasma diamine oxidase in health and intestinal disease.

In animals, the distribution of the enzyme diamine oxidase is confined, almost exclusively, to the small bowel mucosa. In humans, plasma diamine oxidase is at or below assay detection limits but can be liberated into the circulation from binding sites in the intestine by i.v. heparin. Therefore, the authors wished to see if diamine oxidase could be released by a low and safe dose of heparin (5000 U) and if the resultant area under the concentration-time curve would provide a noninvasive marker of segmental intestinal disease. In 17 control subjects, the mean area under the curve (following administration of 5000 U i.v. heparin) was 35.9 +/- 5.0 (SEM) mU.L-1.2 h-1; in 6 individuals studied on two separate occasions, postheparin plasma diamine oxidase profiles were reproducible (r = 0.98; p less than 0.001). The longitudinal distribution of diamine oxidase in the gastrointestinal tract, measured in 12 gastric, 16 jejunal, 6 ileal, and 18 colonic biopsies, was similar in humans to that found in animals. In patients with normal peroral biopsies, there was a linear relationship between jejunal mucosal and postheparin plasma diamine oxidase activities (r = 0.84; p less than 0.01). The areas under the curve in controls were then compared with those in patients with segmental intestinal diseases: 21 with ileal disease with or without colonic Crohn's disease (10 unoperated and 11 with ileal resection), 7 with non-Crohn's ileal resection, 8 with ulcerative colitis, 10 with untreated and 7 with treated celiac disease (6 studied before and after a gluten-free diet), and 5 studied during total parenteral nutrition and again after resumption of oral feeding. The results in the 18 ileectomized patients were subdivided into those with major (arbitrarily greater than 75 cm) and minor (less than 75 cm) resections. Areas under the curve were markedly reduced in nonresected Crohn's patients (6.0 +/- 1.79 mU.L-1.2 h-1; p less than 0.001 vs. controls), correlating inversely, in a first-order relationship, with disease activity (r = 0.82; p less than 0.001) and returning toward normal in 4 patients achieving disease remission. Low areas under the curve in total parenteral nutrition patients (4.5 +/- 0.9; p less than 0.001) were also reversible on resumption of oral feeding. However, areas under the curve were not significantly lower in patients with limited ileal resection (less than or equal to 75 cm), with celiac disease (untreated and treated), or ulcerative colitis than in controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1982

Inhibition of plant and mammalian diamine oxidases by hydrazine and guanidine compounds.

1. Pig kidney and pea seedling diamine oxidases have similar sensitivity to methylhydrazine and phenylhydrazine as inhibitors. 2. Inhibition of pig kidney and pea seedling enzymes by hydrazine and guanidine compounds is time dependent. To reveal full inhibitory potency, methylhydrazine and aminoguanidine need longer preincubation with plant diamine oxidase as compared with mammalian diamine oxidase. 3. Impromidine, a known H2 histamine receptor agonist with guanidine and imidazole structures, and aminoguanidine have higher inhibitory activity towards pig kidney enzyme in comparison with the pea seedling one. 4. Impromidine inhibits pig kidney diamine oxidase in a noncompetitive manner. The Ki value is 6.6 muM. 5. The 24 hr dialysis of rat intestinal diamine oxidase preincubated with phenylhydrazine or impromidine only partially recovered the enzymic activities. 6. Impromidine inhibits mouse intestinal diamine oxidase in vivo.

Amine Oxidase (Copper-Containing)↗

PubMed1994

Prostaglandin E and analogs of prostacyclin influencing superoxide production by the human neutrophil NADPH oxidase system.

The effects of prostaglandin (PG) E1 and I2 analogs (OP-41483 and OP-2507) on the superoxide generation of human neutrophil NADPH oxidase (EC 1.6.99.6) in both whole-cell and cell-free systems were investigated. In a whole-cell system, OP-2507 inhibited the superoxide generation by neutrophils exposed to phorbol myristate acetate concentration-dependently through its superoxide-scavenging action. The concentration of the drug required for 50% inhibition of the oxidase (IC50) was 21 microM. In a cell-free system, however, the drug in concentrations of < 100 microM did not inhibit the activation of NADPH oxidase by sodium dodecyl sulfate because of its inactivation by the detergent. Although PGE1 and OP-41483 did not inhibit the superoxide production by stimulated neutrophils in a whole-cell system, they both inhibited the activation of NADPH oxidase in a cell-free system concentration-dependently, with IC50 values of 44 and 170 microM, respectively. In addition, in the cell-free system, the Km value for NADPH of the oxidase was unchanged by PGE1. The results suggest that the PGI2 analog, OP-2507, is a possible superoxide-scavenger and that PGE1 inhibits the NADPH oxidase activation by sodium dodecyl sulfate in a cell-free system concentration-dependently.

Alprostadil↗

PubMed1986

Measurement of NADPH oxidase activity in detergent lysates of human and mouse macrophage monolayers.

An assay to measure NADPH oxidase activity in detergent lysates of macrophage monolayers is described. The addition of a reaction mixture containing appropriate concentrations of disrupting detergents, NADPH as oxidase substrate and cytochrome c as electron acceptor, to macrophages monolayers permits the reliable detection of a superoxide dismutase-sensitive NADPH-dependent cytochrome c reductive activity. This activity is strictly substrate dependent and NADH could not substitute for NADPH. The NADPH-dependent superoxide anion-forming activity (NADPH oxidase) was investigated in different populations of human and mouse macrophages. NADPH oxidase was activated by stimulation of macrophages with phorbol-myristate acetate and activity levels correlated with ability of intact cells to produce superoxide anion. The optimal conditions for assay of NADPH oxidase were investigated and the assay was used to measure the kinetic properties of the NADPH oxidase. The assay permits investigations of the enzymatic basis of oxidative metabolism in macrophages cultivated as adherent cells without any requirements for recovery of the cells in suspension and subcellular fractionation.

Animals↗

PubMed1994

Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.

Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

Alprostadil↗

PubMed1984

Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate.

The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate (TM). TM strongly inhibited the oxidase activity of five copper oxidase with I50% values in the 1-5 microM range. The mechanism of the TM effect on the copper oxidase, ceruloplasmin (Cp) (E.C. 1.16.3.1), was studied in detail. In Vmax vs. E plots, TM gave parallel data suggesting irreversibility but a large number of TM molecules per Cp were required. The inhibition of Cp by TM could not be reversed by dialysis. Isolation of TM-inhibited Cp on Sephadex G-10 did not yield any active Cp molecules. Cu(II) did not restore any inhibited oxidase activity. Gel electrophoresis supported the covalent binding of Cp by TM without any extensive change in protein structure. EPR results confirmed that Cu(II) is reduced to Cu(I) after reaction with TM. However, the Mo(VI) in MoS4(2-) did not change in oxidation number. Analysis of the TM-Cp compound accounted for all six Cu atoms as found in native Cp. The data suggest the covalent binding of sulfide to Cp copper. TM also inhibited the activity of ascorbate oxidase, cytochrome oxidase, superoxide dismutase, and tyrosinase. However, no inhibition of carbonic anhydrase, a zinc enzyme, was observed at 1 mM TM.

Animals↗

PubMed1994

Purification and active-site characterization of equine plasma amine oxidase.

An improved purification scheme for an amine oxidase from equine plasma (EPAO), a nonruminant source, is described and the protein's active-site is characterized. EPAO is dimeric and contains one Type-2 Cu(II) ion per monomer. The EPAO Cu(II) site is spectroscopically very similar to the Cu(II) sites in other amine oxidases. Unlike the extensively investigated nonruminant amine oxidase from porcine plasma, EPAO does not display half-of-the-sites reactivity; titrations with p-nitrophenylhydrazine and phenylhydrazine indicate two active cofactors per dimer. This cofactor is determined to be the same as that of other copper-containing amine oxidases, 6-hydroxydopa quinone (topa quinone). Upon anaerobic reduction with substrate at ambient temperature, the EPR spectrum of EPAO exhibits a sharp signal at g congruent to 2, attributable to the topa semiquinone. Equine plasma amine oxidase possesses novel in vitro substrate specificity; while other mammalian amine oxidases oxidize norepinephrine only slowly or not at all, EPAO displays significant activity toward this biogenic amine.

Amine Oxidase (Copper-Containing)↗

PubMed1993

Induction of peroxisomal acyl-CoA oxidase by 3-thia fatty acid, in hepatoma cells and hepatocytes in culture is modified by dexamethasone and insulin.

The effects of tetradecylthioacetic acid (TTA) (50 microM), dexamethasone (0.25 microM) and insulin (0.4 microM) on induction of peroxisomal acyl-CoA oxidase activity and mRNA levels were studied in short term cultures of Morris 7800C1 and MH1C1 hepatoma cells and of rat hepatocytes. Dexamethasone and TTA resulted in parallel increases in the enzyme activity and the steady state mRNA content in the hepatoma cells. Combination of dexamethasone and TTA resulted in a synergistic and parallel stimulation of both the enzyme activity and the mRNA levels up to 11-12-fold and maximal changes were observed after 14 days of treatment. Semiquantitative immunoblot analyses of acyl-CoA oxidase were in concordance with enzyme and mRNA results. Insulin counteracted the inductive effects of dexamethasone and TTA on all parameters. The half-life of the acyl-CoA oxidase mRNA increased after treatment with the 3-thia fatty acid (t1/2 = 10.0 h +/- 0.4) compared to control (t1/2 = 5.9 h +/- 0.3). However, in combination with dexamethasone there was no further increase in the mRNA stability (t1/2 = 8.0 h +/- 0.3). Southern blot analysis did not reveal any changes on the oxidase gene level in any treatment group. TTA alone or in combination with dexamethasone did not affect the expression of either the glucocorticoid receptor or the peroxisomal proliferator acting receptor (PPAR) steady state mRNA levels. In cultured hepatocytes the acyl-CoA oxidase was modified in similar manner by these treatments, but the changes were less marked. We suggest that the changes in peroxisomal acyl-CoA oxidase activity in hepatoma cells are due to a major effect on the level of mRNA, involving both transcriptional effects and message stabilization.

Acyl-CoA Oxidase↗

PubMed1983

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum.

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.

Aspergillus niger↗

PubMed1991

Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger.

Glucose oxidase from Aspergillus niger was purified to homogeneity by hydrophobic interaction and ion-exchange chromatography. Approx. 95% of the carbohydrate moiety was cleaved from the protein by incubation of glucose oxidase with endoglycosidase H and alpha-mannosidase. Cleavage of the carbohydrate moiety effected a 24-30% decrease in the molecular weight and a reduction in the number of isoforms of glucose oxidase. No significant changes were observed in the circular dichroism spectra of the deglycosylated enzyme. Other properties, such as thermal stability, pH and temperature optima of glucose oxidase activity and substrate specificity were not affected. However, removal of the carbohydrate moiety marginally affected the kinetics of glucose oxidation and stability at low pH. From these results it appears that the carbohydrate chain of glucose oxidase does not contribute significantly to the structure, stability and activity of glucose oxidase.

Amino Acid Sequence↗