In vitro 6xHis-CBU0823 activity assays for ME (A), LDH (B) and MDH (C) function, including the influence of varied pH (D), and substituted cofactors (E and F).
2021
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Zugriff:
(A) ME activity was observed for 20 μg 6xHis-CBU0823 in the ME standard assay containing 3 mM malate, 5 mM NAD + , 1 mM MnCl 2 . 28.8 μg 6xHis-oMLE and 6xHis-mock purification negative control had no activity. (B) LDH activity was not detected for 20 μg 6xHis-CBU0823 in the LDH standard assay containing 2 mM pyruvate and 0.5 mM NADH, with or without the addition of MnCl 2 . 28.8 μg 6xHis-oMLE and 6xHis-mock purification also failed to show activity, while 0.1 units commercial LDH enzyme had measurable performance in standard conditions. (C) MDH activity was measured for 20 μg 6xHis-CBU0823 in the MDH standard assay containing 2 mM OAA, 0.5 mM NADH, with improved action on addition of 1 mM MnCl 2 . 0.013 units commercial pig heart mitochondrial MDH and 1 μg GST-CBU1241 had efficient activity in the assay and 6xHis-mock purification had no detectable activity. (D) Enzyme activity relative to pH 7.4 over a range of pHs, measured using ME standard assay containing 2 μg 6xHis-CBU0823. Activity was measured using increase or decrease of light absorbance at 340 nm, correlating to NADH oxidation and NAD + reduction respectively during enzyme activity. (E) Removal of MnCl 2 and/or substitution with NADP + cofactor from the standard ME assay reduced 6xHis-CBU0823 activity. Conditions with significantly less activity are indicated. (F) Replacement of the MnCl 2 cofactor with 1mM CaCl 2 , CuCl 2 , MgCl 2 , or ZnSO 4 achieved varying levels of 6xHis-CBU0823 function. Those with significantly lower cofactor facility compared to 1 mM MnCl 2 on ordinary one-way ANOVA are indicated ( p < 0.05). Error bars represent standard deviation around the mean from at least three independent experiments.
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In vitro 6xHis-CBU0823 activity assays for ME (A), LDH (B) and MDH (C) function, including the influence of varied pH (D), and substituted cofactors (E and F).
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Autor/in / Beteiligte Person: | Janine Hofmann (11273418) ; Mebratu A. Bitew (11273421) ; Miku Kuba (11273424) ; David P. De Souza (2793088) ; Hayley J. Newton (8692020) ; Fiona M. Sansom (8893082) |
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Veröffentlichung: | 2021 |
Medientyp: | Bild |
DOI: | 10.1371/journal.pone.0255925.g005 |
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