Direct Measurement of the Binding of Labeled Sugars to the Lactose Permease M Protein

Previous studies of the interaction of sugars with the membrane protein (M protein) component of the lactose permease system have employed an indirect procedure based on the protection of a reactive cysteine in the M protein against attack by labeled N-ethylmaleimide when certain sugars are bound. A method has now been devised for measuring the binding of labeled sugars to M protein directly, avoiding the use of N-ethylmaleimide. The particulate, membrane-containing fraction is equilibrated with 50 µm β-[3H]galactosyl-1-thio-d-β-galactoside (thiodigalactoside), in phosphate buffer containing 32P equal to the total count of 3H. The membrane fragments are then sedimented by high speed centrifugation, and, after careful removal of the supernatant (but without washing), the pellet is resuspended in a detergent solution and counted. The absorption of thiodigalactoside to the membrane fragments is revealed by an increment in the ratio of 3H to 32P. Control experiments show that 32P equilibrates with at least 95% of the total aqueous compartment of the pellet. The increment in the ratio of 3H: 32P is, therefore, not the result of the preferential filling of cell-free vesicles by the radioactive sugar. This binding reaction is not affected by energy poisons such as azide. Membranes from cells that have not been induced for the lac system and thus contain no M protein have no specific binding sites for thiodigalactoside as defined by this assay. The specific binding of thiodigalactoside involves a limited number of saturable binding sites. The value for half-saturation with thiodigalactoside is about 67 µm, in excellent agreement with previous studies of the affinity of the M protein for thiodigalactoside obtained with the indirect N-ethylmaleimide procedure. The site for binding thiodigalactoside (Site II) has also been found to have a surprising affinity for certain α-galactosides, such as p-nitrophenyl-α-galactoside, for which the Kd is about 7 µm. Other properties of the system are described. A procedure is described for the preparation and purification of tritiated thiodigalactoside.