Reconstitution of the Melibiose Carrier of Escherichia coli

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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 Km 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.