To explore how to calculate the effect of solanine on the Michaelis constant and the maximum reaction rate of NAT using the double reciprocal method. High performance liquid chromatography (HPLCP) was used, with 2-AF as substrate and its concentration as substrate concentration, and the rate at which 2-AF is acetylated into 2-AAF in intact HepG2 cells or in the cytoplasm of HepG2 cells as the reaction rate. The double reciprocal plot was made, with 1/S (reciprocal of the concentration of the substrate 2-AF) plotted against 1/V (reciprocal of the reaction rate), to yield a regression equation for calculating Km and Vmax. Studies on enzymetic kinetics have shown that, with intact HepG2 cells, the Km and Vmax for the negative control are 2.37×10-3±8.37×10-5 mM and 9.16×10-4±7.54×10-5nmol/106 cells, respectively, and that the Km and Vmax for the solanine group are 2.22×10-3±9.05×10-5mM and 5.14×10-4±3.72×10-5nmol/106 cells, respectively. For the cytoplasm of HepG2 cells, the Km and Vmax for the negative control are 8.95×10-3±2.61×10-4 mM and 2.55×10-6±1.92×10-8nmol/min·mg protein, respectively, and the Km and Vmax of the solanine group are 9.48×10-3±3.63×10-4mM and 2.43×10-6±1.32×10-8 nmol/min·mg protein, respectively. Statistical analysis showed that, for both intact HepG2 cells and cytoplasm of HepG2 cells, Km does not differ significantly between the negative control and the solanine groups, but Vmax does differ significantly for these groups (p<0.001 for the intact cells, p<0.05 for the cytoplasm). Solanine is a non-competitive inhibitor for the 2-AF substrate of NAT, and the double reciprocal method is a convenient and accurate method for calculating the constants of NAT kinetics.
Substrate concentration dependence of apparent maximum reaction rate and Michaelis constant observed in immobilized enzyme reactions.
