The enzyme-substrate compounds of bacterial catalase and peroxides

Chance, Britton; Herbert, D.

doi:10.1042/bj0460402

Cited by 156 publications

(54 citation statements)

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“…The rabbit Dpb4p polyclonal antibodies were raised against Dpb4p that was overexpressed and purified from E. coli. Aliquots of each fraction were also removed for spectrophotometric assay of catalase activity (23). Whole cell extracts from wild-type yeast were prepared as previously described, and cleared lysate was loaded onto the 15-30% glycerol gradient in buffer C 400 .…”

Section: Methodsmentioning

confidence: 99%

The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

Chilkova¹,

Jonsson²,

Johansson³

2003

Journal of Biological Chemistry

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DNA polymerase ⑀ (Pol ⑀) from Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol ⑀ have been cumbersome due to protease sensitivity and the limited amounts of Pol ⑀ in cells. We have developed a protocol for overexpression and purification of Pol ⑀ from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol ⑀ was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 Å), a molecular mass for Pol ⑀ of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol ⑀. Thus, both DNA polymerase ␦ and Pol ⑀ are purified as monomeric complexes, in agreement with accumulating evidence that Pol ␦ and Pol ⑀ are located on opposite strands of the eukaryotic replication fork.Pioneering studies in the SV40 DNA replication system by several research groups resulted in the reconstitution of the SV40 replication fork with mammalian proteins in vitro (1). This system showed that DNA polymerase ␣ is required for priming the DNA since it is associated with primase activity. Only DNA polymerase ␦ (Pol ␦) 1 was required to replicate both leading and lagging strand, leaving another essential eukaryotic DNA polymerase, DNA polymerase ⑀ (Pol ⑀), without a specific function. However, there are several lines of evidence for the presence of Pol ⑀ at the eukaryotic replication fork. Evidence for a physical interaction came from cross-linking

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Section: Methodsmentioning

confidence: 99%

The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

Chilkova¹,

Jonsson²,

Johansson³

2003

Journal of Biological Chemistry

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“…CAT was estimated using a modification of the method described by Chance and Herbert (1950). Briefly, the reaction mixture contained 200 lg of tissue homogenate and 19.6 mM H 2 O 2 in 2 mL of 10 mM potassium phosphate buffer (pH 7.4).…”

Section: Evaluation Of Scavenging Enzymesmentioning

confidence: 99%

Anti-rheumatic activity ofAnanas comosusfruit peel extract in a complete Freund’s adjuvant rat model

Kargutkar

Brijesh

2016

Pharmaceutical Biology

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Context: Rheumatoid arthritis is a chronic, autoimmune and systemic inflammatory disease, which targets synovial joints leading to joint destruction mediated in part by migration of inflammatory cells into the synovial tissue. Objective: The present study evaluates the anti-rheumatic effect of a methanol extract of Ananas comosus (L.) Merr. (Bromeliaceae) peel in rats. Materials and methods: Anti-rheumatic activity of crude extract of peels of A. comosus in complete Freund's induced arthritis model in rats was studied at doses of 50, 100, 250 and 500 mg/kg b.w. for 21 days. Parameters such as paw size, levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), C-reactive proteins (CRP) and prostaglandins (PGE 2 ) were analysed. Results: Oral administration of the extract significantly reduced the swelling in the paw of rats (EC 50 65.1 ± 2.95 mg/kg b.w.) with a maximal inhibition of 77.01 ± 10.53% on 21st day at 500 mg/kg b.w. The extract also significantly reduced the levels of SOD, CAT and GPx in liver (EC 50 26.84 ± 16.37, 68.37 ± 19.22, 106.54 ± 34.81 mg/kg b.w., respectively), kidney (EC 50 261.75 ± 81.5, 176.38 ± 8.08, 14.32 ± 6.64, mg/kg b.w., respectively) and spleen (EC 50 152.14 ± 39.57, 83.97 ± 14.6, 47.1 ± 10.45 mg/kg b.w., respectively); and CRP (EC 50 36.37 ± 12.4 mg/kg b.w.) and PGE 2 (EC 50 191.06 ± 71.54 mg/kg b.w.) in tissue homogenate and serum, respectively, at 500 mg/kg b.w. as compared to arthritic control group. Discussion and conclusion: These results suggest that A. comosus fruit peel extract exerts anti-rheumatic activity.ARTICLE HISTORY

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“…It has been shown that the catalase Km values derived by double-reciprocal plots can be influenced by the formation of inactive catalase peroxide complexes (1). This cannot explain the enhancement effect, nor does it detract from the inhibitor-protective agent dual effect as an explanation of the unusual double reciprocal plots.…”

Section: Resultsmentioning

confidence: 91%

“…Such inactivation has, in fact, been well-documented (1). If the inhibitor acts by reducing the efficiency of a given enzyme molecule (as opposed to an all-ornone competitive or a dead-end type inhibition) and if the inhibitor also serves a protective function, one can envision a point where the protective effect exceeds the inhibitory effects.…”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of a Catalase Inhibitor from Maize

Sorenson¹,

Scandalios²

1980

Plant Physiol.

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Some biochemical properties of the catalase inhibitor purified from maize scutella are described. The inhibitor is heat-labile and its activity is destroyed by trypsin, indicating that it is a protein. It does not appear to be a lectin nor does the inhibition involve proteolysis. The active inhibitor is a dimer with each subunit having a molecular weight of 5600 as determined by sodium dodecyl sulfate electrophoresis. A kinetic analysis performed in the presence of increasing levels of inhibitor gave unusual Lineweaver-Burk patterns. Possible explanations for these patterns are discussed. The inhibitor is active against all catalases tested from a wide variety of organisms.A substance in maize scutella which inhibits the enzyme catalase (H202:H202 oxidoreductase, EC 1.11.1.6) has been reported; this substance has been purified by affmity chromatography using immobilized catalase (16). The activity of the inhibitor varies inversely with catalase activity in the scutellum of the germinating seed (17) and constitutes one of several mechanisms regulating catalase activity in that tissue (10,12,18). This report describes some of the biochemical properties of this inhibitor. MATERIALS AND METHODSMaterial. Seeds from the highly inbred lines W64A and 229 were used in all studies. Seeds were imbibed in tap water for 12 to 18 h, transferred to plastic trays lined with moist germination papers, and maintained in the dark at 27 C until use. Seedlings were watered daily.Inhibitor Preparation and Assay. The inhibitor was purified by a modification of the affinity chromatography method previously described (16). A scutellar extract was passed through a column containing bovine liver catalase-Sepharose. Following extensive washing with 0.5 M galactose and 0.1 M NaCl, the bound inhibitor was eluted with 1 M NaCl. The galactose wash removed several proteins which apparently adsorbed to the Sepharose matrix. The I M NaCl wash was dialyzed against 10 mM Tris-HCl buffer (pH 7.4) and was stored at 4 C for up to I week. Such preparations lost less than 15% of their original activity during that storage period. The inhibitor was assayed against partially purified maize catalase as previously described (17).Trypsin Digestion. to conduct the digestion. Following incubation, the trypsin was removed by centrifugation at l0,OOOg and the aliquots were assayed for inhibitory activity.After the initial inhibitor action period (approximately 5 min; Fig. 3), no further decrease in catalase activity was observed. This suggests that the centrifugation effectively removed the insolubilized trypsin. Incubation of catalase with insolubilized trypsin under the inhibitor assay times and conditions did not result in a significant decrease in catalase activity.Mol Wt Determination. The mol wt of the native protein was determined by electrophoresis of purified inhibitor under nondenaturing conditions in gels composed of 9.5, 10.0, 10.5, and 11.0% acrylamide as described by Hendrick and Smith (3). The inhibitor does not stain well on gels (see "Dis...

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The enzyme-substrate compounds of bacterial catalase and peroxides

Cited by 156 publications

References 2 publications

The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

Anti-rheumatic activity ofAnanas comosusfruit peel extract in a complete Freund’s adjuvant rat model

Biochemical Characterization of a Catalase Inhibitor from Maize

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