Properties of membrane-associated sialyltransferase of Escherichia coli.

Membrane-associated sialyltransferase complexes in Escherichia coli K-235 can be dissociated by lipid deletion and reassembled by the addition of undecaprenyl phosphate, a unique membrane-bound lipid coenzyme. Following disruption of the cells by pressure disintegration and centrifugal fractionation, the sialyltransferase activity is assocatied with both a "particulate" and "soluble" complex. Kinetic studies as well as sugar nucleotide, metal ion, pH, ammonium sulfate, and thiol reagent requirements showed these two complexes contained functionally identical enzymatic activities. Isopycnic sucrose density gradient centrifugation studies carried out on unfractionated total membranes established that these sialytransferase activities were associated with membrane hybrids composed of different relative amounts of inner and outer membranes. Enzyme localization studies employing DPNH oxidase, a marker for the inner membrane, and relative phospholipid to protein composition determinations in the two complexes, provided added support for this conclusion. Sialyl polymer synthesis was not dependent on the incorporation of other monosaccharides and had no demonstrable metal ion requirement. Kinetic studies showed that the Km for cytidine 5-monophospho-N-acetylneuraminic acid in intact soluble and particulate enzyme preparations was 8.1 times 10-5M and 9.2 times 105M, respectively. Similarly, both enzyme complexes had nearly identical Vmax values. Following reassembly of delipidated enzyme preparations, however, there was a 10-fold increase in the Km value for the particulate enzyme and a 3-fold increase for the soluble enzyme. This increase was accompanied by an increase of approximately the same magnitude in the Vmax values. Since the lipid coenzyme was limiting in intact enzyme preparations, the increase in Vmax reflected an increase in the concentration of the active lipid in reconstituted complexes. Sialyl polymer synthesis in intact membrane preparations was stimulated by the exogenous addition of lipid. Insertion of the carrier lipid was dependent on temperature. At 37 degrees, a 120% increase in sialytransferase activity was observed while only a 35% increase was observed at 30 percent. At 20 degrees, no stimulation occurred. Fluidity of the lipid phase is apparently required for proper function of this membraneassociated enzyme complex. Thus, at 20 degrees, a temperature below the membrane lipid transition temperature, the lipids are relatively immobile.