Fig. 3

Effects of temperature and ionic strength on WS activity of Thraustochytrium roseum WS/DGAT proteins TrWSD4 and TrWSD5. The effect of temperature on the relative WS activity of TrWSD4 (a) and TrWSD5 (b) was observed. WS activity was determined with the substrates of 90 μM palmitoyl-CoA and 100 μM hexadecanol. The relative WS activity was calculated at different temperatures by taking the WS activity at 37 °C as 100%. The influence of temperature on the values of kinetic parameters K _m and V _max for TrWSD4- (c) and TrWSD5- (d) catalyzed reaction was observed. The value of fold change was calculated at different temperatures by taking the value at 17 °C as 1.0. Reactions were performed in mixtures including 25 mM sodium phosphate buffer (pH 7.4) and 1 mg/mL DTNB. The effect of ionic strength on the WS activity of TrWSD4 (e) and TrWSD5 (f) was observed at the reaction temperature of 37 °C. WS activity was determined with the substrates of 90 μM decanoyl-CoA and 100 μM hexadecanol for a wide range of NaCl concentrations relative to the reaction without addition of NaCl. Reactions were performed at 37 °C in mixtures including 25 mM sodium phosphate buffer (pH 7.4), 1 mg/mL DTNB and different concentrations of NaCl. WS activity values are averages of a representative experiment performed in triplicate and error bars correspond to one standard deviation (S.D.)