Calculation of the Effect of Solanine on the Michaelis Constant and the Maximum Reaction Rate of NAT

To explore how to calculate the effect of solanine on the Michaelis constant and the maximum reaction rate of NAT, high performance liquid chromatography (HPLC) was used, with 2-AF as substrate, 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 K_m and V_max. With intact HepG₂ cells, the K_m and V_max for the negative control are 2.37times10^-3plusmn8.37times10^-5 mM and 9.16times10^-4plusmn7.54times10^-5 nmol/10⁶ cells, respectively, and that the K_m and V_max for the solanine group are 2.22times10^-3plusmn9.05times10^-5 mM and 5.14times10^-4plusmn3.72times10^-5 nmol/10⁶ cells, respectively. For the cytoplasm of HepG₂ cells, the K_m and V_max for the negative control are 8.95times10^-3plusmn2.61times10^-4 mM and 2.55times10^-6plusmn1.92times10^-8 nmol/ minmiddotmg protein, respectively, and the K_m and V_max of the solanine group are 9.48times10^-3plusmn3.63times10^-4 Mm and 2.43times10^-6plusmn1.32times10^-8 nmol/minmiddotmg protein. Statistical analysis showed that, for both intact HepG₂ cells and cytoplasm of HepG₂ cells, K_m does not differ significantly between the negative control and the solanine groups, but V_max does differ significantly for these groups (P<0.05). Solanine is a non-competitive inhibitor for the 2-AF substrate of NAT.