Table 2 Conditions used to measure enzymatic activity.

From: The oxidoreductase activity of Rnf balances redox cofactors during fermentation of glucose to propionate in Prevotella

Enzyme

Reference

Assay componentsa,b

Product measured (wavelength)

Controls

Conditionsc

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12, 1.2.1.13, 1.2.1.59)

After Zheng et al.68

50 mM Tricine-Na (pH 8.4), 10 mM potassium phosphate buffer (pH 7), 2 mM dithiothreitol (DTT), 2 mM MgCl2, 1 mM glyceraldehyde 3-phosphate, 1 µg of cell extract protein, 1 mM NAD sodium salt or NADP disodium salt

Reduced NAD(P) (340 nm)e

Cell extract replaced with water

Aerobic

Malate dehydrogenase (EC 1.1.1.37, 1.1.1.82)

After Zeikus et al.69

50 mM Tris–Cl (pH 7.6), 0.2 mM NADH disodium salt or NADPH tetrasodium salt, 1 µg of cell extract protein, 2 mM oxaloacetic acid

Reduced NAD(P) (340 nm)e

Cell extract replaced with water

Aerobic

Pyruvate dehydrogenase (EC 1.2.4.1)

This study

50 mM Tris–Cl (pH 7.6), 10 mM MgCl2, 4 mM DTT, 0.2 mM CoA lithium salt, 0.1 mM thiamine pyrophosphate, 4 U/mL phosphotransacetylase, 2 mM NAD sodium salt, 15.1 µg of cell extract or cytoplasmic contents protein, 10 mM sodium pyruvate

Reduced NAD (340 nm)e

Cell extract or cytoplasmic contents replaced with water

Anaerobic

Pyruvate:ferredoxin oxidoreductase (PFOR) (EC 1.2.7.1, 1.2.7.11)

After Zheng et al.68

50 mM Tris–Cl (pH 7.6), 10 mM MgCl2, 4 mM DTT, 0.2 mM CoA lithium salt, 30 µM ferredoxin, 0.1 mM thiamine pyrophosphate, 4 U/mL phosphotransacetylase, 15.1 µg of cell extract or cytoplasmic contents protein, 10 mM sodium pyruvate

Reduced ferredoxin (430 nm)f

Cell extract or cytoplasmic contents replaced with water

Anaerobic

Rnf (Ferredoxin:NAD+ oxidoreductase [Na+-transporting]) (EC 7.2.1.2)

After Zheng et al.68

50 mM Tris–Cl (pH 7.6), 10 mM MgCl2, 4 mM DTT, 10 mM NaCl, 80 µg of cell membrane protein (or 40 µg of solubilized cell membrane protein), reduced ferredoxin-regenerating system (0.2 mM CoA lithium salt, 30 µM ferredoxin, 0.1 mM thiamine pyrophosphate, 4 U/mL phosphotransacetylase, 36.2 µg of cytoplasmic contents protein, 10 mM sodium pyruvate), 2 mM NAD sodium salt

Reduced NAD (340 nm)e

Cell membrane replaced with water

Anaerobic

Nqr (NADH:ubiquinone reductase [Na+-transporting]) (EC 7.2.1.1)

This study

100 mM potassium phosphate (pH 6), 100 mM NaCl, 4 mM DTT, 0.4 mM NADH disodium salt, 40 µg of solubilized cell membrane protein or 80 µg of cell membrane protein

Reduced NAD (340 nm)e

Cell membrane replaced with water

Anaerobic

Fumarate reductase/succinate dehydrogenase (EC 4.2.1.2)

After Asanuma and Hino70

100 mM potassium phosphate (pH 6), 100 mM NaCl, 4 mM DTT, 0.4 mM NADH disodium salt, 40 µg of solubilized cell membrane protein or 80 µg of cell membrane protein, 5 mM disodium fumarate

Reduced NAD (340 nm)e

Fumarate and cell membrane replaced with water

Anaerobic

ATPase (EC 7.1.2.2)

After Schoelmerich et al.29

100 mM Tris–Cl (pH 7.4), 5 mM MgCl2, 6.25 µg of cell membrane protein or solubilized cell membrane protein, 3.6 mM ATP-DiTrisd

Phosphomolybdate (335 nm)g

None

Aerobic

  1. aThe components are listed in the order added (with the last component added to initiate the reaction).
  2. bSource: NADH disodium salt, Sigma N8129; NADPH tetrasodium salt, Calbiochem 481,973; oxaloacetic acid, Sigma O4126; coenzyme A lithium salt, Calbiochem 234,101; ferredoxin, purified from C. pasteurianum 5 according to Schönheit et al.71, phosphotransacetylase, Megazyme E-PTABS; crude pyruvate:ferredoxin oxidoreductase, cytoplasmic contents from same bacterium being assayed for activity.
  3. cAnaerobic conditions were 1 mL assay mix in 1.4 mL glass cuvette (Hellma HL114-10–20) capped with chlorobutyl stopper (DWK Life Sciences W224100-081) under N2 at 37 °C; aerobic conditions were 0.2 mL assay mix in 96-well plates at room temperature; aerobic conditions for ATPase assay were 0.1 mL assay mix in 1.5 mL tube at 37 °C.
  4. dMix was incubated for 0, 4, 8, and 12 min and reaction terminated by adding 14.3 µL of 30% (w/v) trichloroacetic acid.
  5. eExtinction coefficient of 6,200 M−1 cm−1, see reference68.
  6. fExtinction coefficient of 13,100 M−1 cm−1, see reference72.
  7. gPhosphomolybdate was formed by adding 90 µL supernatant with 450 µL of AAM-reagent73 and incubating for 10 min at room temperature; phosphate was the standard.
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