CXCVIII.—Universal buffer solutions and the dissociation constant of veronal

Britton, H. T. S.; Robinson, R. A.

doi:10.1039/jr9310001456

Cited by 677 publications

(356 citation statements)

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“…The inhibitors used in the experiment were 5 mM 1,10-phenanthroline to inhibit metalloendoproteases, 2 mM pepstatin to inhibit aspartic proteases, 10 mM phenylmethylsulfonyl fluoride to inhibit serine proteases and 10 mM trans-epoxysuccinyl-Lleucylamido(4-guanidino)butane to inhibit cysteine proteases. In the assay, 4 ml of the diluted fecal fluid was mixed with 1 ml of inhibitor or distilled demineralized water and incubated on ice for 1 h. Next, 35 ml of 0.1 M Britton Robinson buffer (Britton and Robinson, 1931) with 2% azoalbumin was added and the mix was incubated at 30 1C for 1 h. For the blank control (not incubated), the azoalbumin mix was added at the last moment without incubation to prevent any protease activity to occur. To stop the activity of the proteases, 120 ml of 10% trichloroacetic acid was added, after which the mix was vortexed and incubated for 15 min before centrifugation for 5 min at 8000 g. The supernatant (86 ml) was added to 100 ml of freshly prepared 1 M NaOH on a 96-well cell culture plate.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

et al. 2014

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The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5-7, but the aspartic proteases were inactive across a pH range of 3-10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.

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Section: Enzyme Assaysmentioning

confidence: 99%

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

et al. 2014

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“…The 0.04 mol L -1 Britton-Robinson (BR) buffer solutions, used as the supporting electrolyte, were prepared as described in the literature and the pH was adjusted to the desired value by adding appropriate amounts of 0.2 mol L -1 NaOH stock solution. 19 All reagents used were analytical grade.…”

Section: Equipments and Reagentsmentioning

confidence: 99%

Direct electrochemical analysis of dexamethasone endocrine disruptor in raw natural waters

Oliveira

Ribeiro

Nascimento

et al. 2012

J. Braz. Chem. Soc.

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Este trabalho descreve uma metodologia eletroanalítica, utilizando voltametria adsortiva de onda quadrada, que foi aplicada com êxito na determinação direta de resíduos de dexametasona em águas naturais brutas utilizadas no abastecimento público do Estado do Ceará, Brasil. Os limites de detecção obtidos variaram entre 7,47 × 10 -9 e 1,80 × 10 -8 mol L -1 para as três matrizes de águas naturais brutas avaliadas. Os valores percentuais médios de recuperação (98,86% ± 0,72), repetibilidade (0,32% ± 0,05) e reprodutibilidade (0,91% ± 0,20) foram significativos, reafirmando a sensibilidade do procedimento. A energia dos orbitais LUMO e as cargas atômicas de Mülliken foram calculadas usando o funcional BLYP/DNP. Os resultados teóricos, aliados aos critérios de diagnóstico da voltametria de onda quadrada, indicam que o mecanismo redox da dexametasona está associado a processos de redução quase-reversível e irreversível dos grupos cetona localizados em C-20 e C-3, respectivamente. This paper describes an electroanalytical methodology using square-wave adsorptive voltammetry, which has been successfully applied for the direct determination of dexamethasone residues in raw natural waters used for the public supply of the Ceará State, Brazil. The obtained detection limits ranged from 7.47 × 10 -9 to 1.80 × 10 -8 mol L -1 for the three matrices of raw natural waters evaluated. High percentages of average recovery (98.86% ± 0.72), repeatability (0.32% ± 0.05) and reproducibility (0.91% ± 0.20) were obtained in these samples, reaffirming the sensitivity of the procedure. Energy of the LUMO orbitals and Mülliken's atomic charges were calculated using the functional BLYP/DNP. The theoretical results allied to the diagnostic criteria of the square-wave voltammetry indicate that the dexamethasone redox mechanism is associated to the quasi-reversible and irreversible reduction process of the ketone groups located at C-20 and C-3, respectively.

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“…The effect of temperature was tested in PC buffer, pH 6.6, at temperatures ranging from 30 uC to 80 uC. The effect of pH on the activity was assayed at 37 uC in a series of BrittonRobinson buffers from pH 3.0 to 9.0 (0.1 M boric acid, 0.1 M acetic acid, 0.1 M phosphoric acid, adjusted to the desired pH with NaOH) (Britton & Robinson, 1931). Thermal stability of the enzymes was estimated by incubating the diluted solution of enzymes in PC buffer, pH 6.6, at 55 uC for AbfA and 70 uC for Abf2.…”

Section: Ip: 54191190102mentioning

confidence: 99%

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

2008

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Bacillus subtilis produces a-L-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and L-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (.250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 6C, with p-nitrophenyl-a-L-arabinofuranoside as substrate, gave an apparent K m of 0.498 mM and 0.421 mM, and V max of 317 U mg "1 and 311 U mg "1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 6C and 60 6C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1A5) linkages of linear a-1,5-L-arabinan and a-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1A2) and (1A3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinosecontaining polysaccharides by B. subtilis.

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CXCVIII.—Universal buffer solutions and the dissociation constant of veronal

Cited by 677 publications

References 0 publications

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

Direct electrochemical analysis of dexamethasone endocrine disruptor in raw natural waters

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

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