Membrane-associated Nucleotide Sugar Reactions

Galactose-diphosphoglycosyl carrier lipid (galactose-PP-GCL) synthetase, a membrane-associated enzyme, catalyzes the first reaction in the biosynthesis of the O antigen of Salmonella typhimurium LT2. The enzyme transfers galactose-1-P from UDP-galactose to phosphoglycosyl carrier lipid (P-GCL) to form GCL-PP-Gal, a lipid-extractable product. Nucleotide exchange, specific for UDP-galactose and UMP, is a sensitive assay for this activity. With isolated active membrane preparations, the extent of the exchange reaction depends on the concentrations of UDP-galactose and UMP rather than the concentration of available endogenous P-GCL. The exchange reaction follows pseudo-first order kinetics, with an apparent Km for UDP-galactose of 2.5 x 10-5 m. The presence of MgCl2 is required for optimal activity. The association of GCL-PP-Gal synthetase and the cell membrane is disrupted by some methods of mechanical cell breakage, and fully active enzyme is released into supernatant fractions of the cell. No activity was detected in the supernatant when lysozyme-EDTA spheroplasts were fractionated. One of the enzymes of cell wall synthesis, phosphoN-acetylmuramyl-pentapeptide translocase, remains membrane-associated regardless of the method of cell breakage. The translocase was shown to be localized on the cytoplasmic membrane using resolved membrane preparations of mechanically broken cells. This method of membrane resolution gave equivocal results for the localization of GCL-PP-Gal synthetase because of the release of a large part of the enzyme activity during cell breakage.