Glycerol kinase assay

Cell extracts were prepared essentially as described before ¹⁷. A 25 mL culture was grown exponentially until OD₆₀₀ = 0.5. Twenty mL of the culture was transferred to a tube precooled on ice water and chloramphenicol was added to give a final concentration of 40 μg/mL. The cells were harvested by centrifugation at 0°C, washed with 40 mL of 1% NaCl, resuspended in 200 μL extraction buffer containing 0.1 M MOPS-NaCl at pH7.0, 1 mM 2-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid, and 2 mM glycerol, and stored at −80°C.

Before the assay, the cell suspension was thawed, disrupted by sonication on ice water for 4 times 5 s at the amplitude of 4 at low mode in a MSE sonicator, centrifuged at 13,600 × g for 30 min at 4°C, and the supernatant was frozen at −80°C. The GlpK activity was stable for one day at −80°C.

The cell extract was thawed immediately before the assay and diluted in the extraction buffer. The reaction was started by adding 40 μL of cell extract to 200 μL of assay buffer, both of which were preincubated at 37°C for 5 min, to give final concentrations of 0.1 M MOPS-NaCl at pH7.0, 0.167 mM 2-mercaptoethanol, 0.167 mM ethylenediaminetetraacetic acid, 2 mM glycerol, 2.5 mM ATP, 13.5 mM MgSO₄, 0.188 mM 4-aminoantipyrine, 2.11 mM N-ethyl-N-(3-sulfopropyl) m-anisidine, 5 U/mL glycerol phosphate oxidase, and 5 U/mL peroxidase. A₅₄₀ was recorded every 6 s. To confirm that the activities were proportional to the concentrations of the cell extracts added to the reaction mixture, the assays were repeated with four different dilutions of the cell extract for each. The GlpK activity was reported as A₅₄₀ per min per mg of total protein. Total amounts of protein in a cell extract were determined by Biuret method ⁴⁸.