반응 #721257

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NAD+

Glycine KOH

MgSO4

→

NADH

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후처리

1. 1
   농축NAD+ was substituted with equal concentrations of NADP+, FMN+, and FAD+, when
2. 2
   workup.ADDITIONThe assay components were mixed in the cuvette
3. 3
   workup.ADDITIONThe reactions were started by addition of 5-40 μg of the purified MDH proteins

실험 절차

Alcohol dehydrogenase activities were measured spectrophotometrically essentially as described previously (Hektor, Kloosterman et al. (2002) Chem 277(49): 46966-46973) and the reaction mixture contained: 100 mM Glycine-KOH pH 9.5 (unless otherwise stated), 5 mM MgSO4, 0.5 mM NAD+ and 500 mM alcohol (methanol, ethanol, propanol, 1,3-propanediol, or butanol). NAD+ was substituted with equal concentrations of NADP+, FMN+, and FAD+, when indicated. The reaction mixture for measurements of formaldehyde reductase activity contained: 50 mM Potassium-phosphate buffer pH=6.7, 0.15 mM NADH, 1 mM DTT and 11.6 mM (0.1-116 mM) formaldehyde. The assay components were mixed in the cuvette and pre-warmed to 45° C., unless otherwise stated. The reactions were started by addition of 5-40 μg of the purified MDH proteins, and the production of NADH was monitored at 340 nm for 4 minutes. One unit of MDH activity was defined as the amount of enzyme needed to produce 1 μmol NADH per minute under the conditions described above. Purified Act (0.1-40 μg) or NudF (20 μg) proteins were added to the reaction mixtures as indicated in the text.

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