Determination of catalase activity by means of the Clark oxygen electrode

Rørth, Mikael; Jensen, P.K.

doi:10.1016/0005-2744(67)90124-6

Cited by 152 publications

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“…Enzyme and Protein Determination-Catalase activity was determined by the method of Rorth and Jensen (27) in a Gilson oxygraph equipped with a Clark electrode. One unit of catalase is defined as the amount that decomposes 1 mol of H 2 O 2 in 1 min in a 60 mM H 2 O 2 solution at pH 7.0 at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Singh

Wiseman

Deemagarn

et al. 2004

Journal of Biological Chemistry

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Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD ؉ and either H 2 O 2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (K m ‫؍‬ 12 M), but the oxidase reaction is slow (k cat ‫؍‬ 0.54 min ؊1 ) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen-and H 2 O 2 -independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD ؉ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD ؉ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD ؉ and INH more efficiently than B. pseudomallei KatG.

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Section: Methodsmentioning

confidence: 99%

Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Singh

Wiseman

Deemagarn

et al. 2004

Journal of Biological Chemistry

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“…Direct catalase activity was determined by measuring the initial rates of O 2 production with a micro-Clark electrode (Del Rio et al, 1977;Chary and Natvig, 1989). The reaction was initiated by injecting protein extracts into a sealed chamber filled with 3 ml of 50 mM Tris-HCl buffer, pH 7.5, and 10 mM hydrogen peroxide (Rorth and Jensen 1967). One unit (U) was defined as the enzyme activity that generates 1 μmol O 2 /min at room temperature, and specific activity was defined as U/mg protein.…”

Section: Strains and Culturementioning

confidence: 99%

Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa

et al. 2007

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Neurospora crassa has four catalase genes-cat-1, cat-2, cat-3, and ctt-1/cat-4. cat-1 and cat-3 encode two fungal-specific large-subunit catalases CAT-1 and CAT-3 normally produced in conidia and growing hyphae, respectively. cat-2 encodes CAT-2 catalase-peroxidase normally produced in conidia. ctt-1 (or cat-4), of which expression was controlled by OS-2 MAP kinase (Noguchi et al., Fungal Genet. Biol. 44,(208)(209)(210)(211)(212)(213)(214)(215)(216)(217)(218), encodes a small-subunit catalase with unknown function. To clarify the contribution of OS-2 on the regulation of CAT-1, CAT-2, and CAT-3, we performed quantitative RT-PCR and in-gel catalase activity analyses. When the hyphae were treated with a fungicide (1 μg/ml fludioxonil) or subjected to an osmotic stress (1 M sorbitol), cat-1 was strongly upregulated and CAT-1 was reasonably induced in the wild-type strain. Interestingly, fludioxonil caused not only the CAT-1 induction but also a remarkable CAT-3 decrease in the wild-type hyphae, implying of an abnormal stimulation of asexual differentiation. These responses were not observed in an os-2 mutant hyphae, indicating an involvement of OS-2 in the cat-1 expression; however, os-2 was dispensable for the production of CAT-1 in conidia. In contrast, the expression of cat-2 was significantly induced by heat shock (45°C) and that of cat-3 was moderately stimulated by an oxidative stress (50 μg/ml methyl viologen) in both the wild-type strain and the os-2 mutant, and corresponding enzyme activities were detected after the treatments. Although basal levels of transcription of cat-1 and cat-3 in an os-2 mutant hyphae were a few-fold lower than in the wild-type hyphae, the os-2 mutant exhibited a considerably lower levels of CAT-3 activity than the wild-type strain. These findings suggest that OS-2 MAP kinase regulated the expression of cat-1 and cat-3 transcriptionally, and probably that of cat-3 posttranscriptionally, even though the presence of another regulatory system for each of these two genes is evident.

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“…Catalase activity was determined with a Clark oxygen electrode by a modification of the procedure of Rorth and Jensen (6). Recovery of catalase activity from the gradient was always greater than 70%, with no quantitative differences between isozymes.…”

Section: Electrophoresismentioning

confidence: 99%

Turnover of Genetically Defined Catalase Isozymes in Maize

Quail

Scandalios

1971

Proc. Natl. Acad. Sci. U.S.A.

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The turnover of two homotetramers of catalase, V4 and Z4, specified by genes at two separate loci, has been studied during germination of maize seed by the techniques of density labeling and starch-gel electrophoresis. Both isozymes were shown to be turning over during this time. However, Z4 accumulates because the rate of synthesis exceeds the rate of degradation, whereas V4 slowly disappears because the rate of degradation exceeds the rate of synthesis. Evidence is also presented that the rate of synthesis of Z4 exceeds that of V4, which suggests that this may be a major factor in the differential expression of the two catalase genes.Maize catalase (H202:H202 oxidoreductase, EC 1.11.1.6) is a tetramer (1). The products of catalase genes at two distinct loci, Ct1 and Ct2, have been detected and shown to be inherited independently of each other (Scandalios, manuscript in preparation). When present together at the same stage of development, the two gene products (subunits) interact to form active tetramers. However, from the time of pollination to just prior to seed maturation, only the product of the Ct1 locus is present in the developing caryopsis of inbred lines homozygous at this locus. Thus, only one isozyme (the homotetramer) is active during this time. The product of the Ct2 locus is first detected in the dry seed and increases dramatically during the first 3 days of germination. The Ct1 gene product, on the other hand, slowly decreases in activity during germination and ultimately disappears. During the time that both classes of subunits are available, five species of active tetramers are detected-two homotetramers and three heterotetramers. Mich. 48823 V4. The polypeptide product of the Ct2 locus is tentatively designated subunit Z; the homotetramer is Z4. In this inbred line, the Z4 isozyme has a greater electrophoretic mobility than the V4 isozyme. MATERIALS AND METHODS Growth and extraction of seedlingsSeeds of maize, inbred line W64A, were germinated between moistened filter papers in Petri dishes in the dark at 240C.The papers were moistened either with 10 mM K'4NO3 in H20 (14N-H20) or with 10 mM (99 atom %) K15N03 in 70% D20 ('5N-D20). In the chase experiments, seeds were germinated for 36 hr in 15N-D20, rinsed for 20 min with several changes of water, and then placed in '4N-H20 for the chase period. Twenty scutella from each treatment were homogenized with sand in a mortar and pestle in 1 ml of 25 mM glycylglycine buffer (pH 7.4) and centrifuged at 20,000 X g for 10 min. The crude supernatant was used directly for electrophoresis. ElectrophoresisStarch-gel electrophoresis and staining for catalase were performed as described (2). For preparative purposes the entire extract from twenty scutella was applied to a single starch gel. After electrophoresis, a thin slice of the gel was stained to facilitate location of the V4 and Z4 isozymes. Strips corresponding to these two isozymes were cut from the remainder of the gel and centrifuged at 30,000 rpm for 30 min in an International B60 ultra...

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Determination of catalase activity by means of the Clark oxygen electrode

Cited by 152 publications

References 0 publications

Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa

Turnover of Genetically Defined Catalase Isozymes in Maize

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