Visualization of catalase on acrylamide gels

Gregory, Eugene M.; Fridovich, Irwin

doi:10.1016/0003-2697(74)90440-0

Cited by 133 publications

(76 citation statements)

References 13 publications

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“…For strains bearing the ubiA420 menA401 alleles, 1 mM p-hydroxybenzoic acid was added to the medium (16) during strain construction to prevent reversion. The katG17::Tn10 and katG14 alleles were confirmed by the absence of the hydroperoxidase I/catalase band on an activity gel (32). The sdhC4::kan r allele was verified by loss of succinate:plumbagin oxidoreductase activity from the membranes of cells grown aerobically in LB medium.…”

Section: Methodsmentioning

confidence: 93%

Mechanism of Superoxide and Hydrogen Peroxide Formation by Fumarate Reductase, Succinate Dehydrogenase, and Aspartate Oxidase

Messner

Imlay

2002

Journal of Biological Chemistry

255

227

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Superoxide dismutases, catalases, and peroxidases are ubiquitous among aerobic and aerotolerant organisms, suggesting that their substrates, superoxide and hydrogen peroxide, are inevitable toxic by-products of aerobic metabolism (1, 2). Considerable progress has been made in identifying the mechanisms by which these species damage cells (2-11). In aerobic environments, mutant organisms that cannot scavenge endogenous O 2. and H 2 O 2 typically grow poorly or die, indicating that these species are formed in potentially toxic doses inside living cells (12, 13). Similar toxicity occurs in wild-type organisms when they are exposed to higher-than-usual levels of oxygen, evidently because these species are formed at elevated rates. Despite this progress, the mechanisms of O 2 . and H 2 O 2 formation are less well understood. Molecular oxygen is a triplet species that can only accept electrons one at a time from potential donors (14). This restriction ensures that oxygen does not spontaneously oxidize most reduced biomolecules, such as NAD(P)H, which are obligate two-electron donors. Instead, enzymes that are competent univalent electron donors are the most likely effectors of inadvertent oxygen reduction. Such enzymes are prominent in electron transport chains. Accordingly, studies in Escherichia coli determined that NADH dehydrogenase II, succinate dehydrogenase, sulfite reductase, and fumarate reductase each formed O 2. and H 2 O 2 when the reduced enzymes were exposed to oxygen (15, 16). These autoxidizing enzymes contain flavins and either iron-sulfur clusters or quinones, all of which are competent at univalent redox reactions. However, in each case the flavin appeared to be the primary site of electron transfer to oxygen. This trend has been noted in other autoxidizing enzymes as well. This may be due to the solvent accessibility of the flavins, which are situated at the protein surface in order to interact with soluble substrates. In contrast, the iron-sulfur clusters are typically buried within polypeptide, and quinones may be sequestered in hydrophobic regions of the proteinmembrane interface where O 2 . formation is disfavored. Interestingly, the rates at which flavoenzymes leak electrons to oxygen vary over several orders of magnitude (15,(17)(18)(19), indicating that additional factors must govern turnover number. We are hopeful that some of these factors might be revealed by study of members of the succinate dehydrogenase/ fumarate reductase family (Fig. 1). These enzymes are structurally and functionally similar to one another (20 -22), and each autoxidizes, albeit at different rates. Succinate dehydrogenase (Sdh), 1 a primary respiratory dehydrogenase, catalyzes electron transfer from succinate to membrane-bound quinone. Fumarate reductase (Frd) catalyzes the opposite reaction in its service as a terminal oxidase during the anaerobic growth of some bacteria and eukarya. The physical structure of the E. coli fumarate reductase has been determined (23), and the pathways of electron movement through both ...

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Section: Methodsmentioning

confidence: 93%

Mechanism of Superoxide and Hydrogen Peroxide Formation by Fumarate Reductase, Succinate Dehydrogenase, and Aspartate Oxidase

Messner

Imlay

2002

Journal of Biological Chemistry

255

227

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“…Cells were disrupted by sonication, and protein concentration was determined by the Bradford assay. In-gel catalase and superoxide dismutase assays were performed as previously described (6,19). Immunoblotting was performed as previously described (38) with polyclonal Mn superoxide dismutase antibody (QED Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Disruption of sitA Compromises Sinorhizobium meliloti for Manganese Uptake Required for Protection against Oxidative Stress

Davies

Walker

2007

J Bacteriol

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During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium meliloti must overcome an oxidative burst produced by the plant in order for proper symbiotic development to continue. While identifying mutants defective in symbiosis and oxidative stress defense, we isolated a mutant with a transposon insertion mutation of sitA, which encodes the periplasmic binding protein of the putative iron/ manganese ABC transporter SitABCD. Disruption of sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide and superoxide. Disruption of sitA leads to elevated catalase activity and a severe decrease in superoxide dismutase B (SodB) activity and protein level. The decrease in SodB level strongly correlates with the superoxide sensitivity of the sitA mutant. We demonstrate that all free-living phenotypes of the sitA mutant can be rescued by the addition of exogenous manganese but not iron, a result that strongly implies that SitABCD plays an important role in manganese uptake in S. meliloti.

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“…A Bradford assay (Bio-Rad) was used to quantify protein in the resulting supernatant with BSA used as the standard. Total protein (75 pg) was resolved on a 7.5% native polyacrylamide gel with a 3.5% stacking gel and stained for catalase activity, as previously described (Gregory and Fridovich, 1974).…”

Section: Lsozyme Cel Analysismentioning

confidence: 99%

Catalase Is Encoded by a Multigene Family in Arabidopsis thaliana (L.) Heynh

et al. 1996

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The catalase multigene family in Arabidopsis includes three genes encoding individual subunits that associate to form at least six isozymes that are readily resolved by nondenaturing gel electrophoresis. CATl and CAT3map to chromosome 1 , and CAT2maps to chromosome 4. The nucleotide sequences of the three coding regions are 70 to 72% identical. The amino acid sequences of the three catalase subunits are 75 to 84% identical and 87 to 94% similar, considering conservative substitutions. Both the individual isozymes and the individual subunit mRNAs show distinct patterns of spatial (organ-specific) expression. Six isozymes are detected in flowers and leaves and two are seen in roots. Similarly, mRNA abundance of the three genes varies among organs. All three mRNAs are highly expressed in bolts, and CAT2 and CAT3 are highly expressed in leaves.

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Visualization of catalase on acrylamide gels

Cited by 133 publications

References 13 publications

Mechanism of Superoxide and Hydrogen Peroxide Formation by Fumarate Reductase, Succinate Dehydrogenase, and Aspartate Oxidase

Mechanism of Superoxide and Hydrogen Peroxide Formation by Fumarate Reductase, Succinate Dehydrogenase, and Aspartate Oxidase

Disruption of sitA Compromises Sinorhizobium meliloti for Manganese Uptake Required for Protection against Oxidative Stress

Catalase Is Encoded by a Multigene Family in Arabidopsis thaliana (L.) Heynh

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