Reconstitution of the Melibiose Carrier of Escherichia coli

Abstract

Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several α-and β-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [³H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na⁺ the K_m for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-β-galactoside and p-nitrophenyl-α-galacto-side were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhy-drazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.