Influence of "energization" on the binding of M protein with p-nitrophenyl alpha-D-galactopyranoside

A specific binding of p-nitrophenyl alpha-D-galactopyranoside has been measured by flow dialysis with Escherichia coli ML 308225 membrane vesicles containing the lac carrier protein. The number of binding sites, 0.45 nmol/mg of membrane protein, remains unchanged in the presence or absence of energy. On the other hand, "energization" increases the M protein affinity for p-nitrophenyl alpha-D-galactopyranoside. The dissociation constant (Kd) is 4 and 21 microM in the presence and absence, respectively, of D-lactate. The same energization effects are found with E. coli A3245 membrane vesicles. p-Nitrophenyl alpha-D-galactopyranoside can be used as a substrate to study energization effect on binding to the lactose permease M protein. These results corroborate observations that energy increases the lac carrier protein affinity for its substrate, and they also confirm the concentration of the M protein, which corresponds to 1.4% of the membrane protein.