A rapid, high-yield procedure has been developed for the purification of HPr from the Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. During this procedure, the protein copurifies with a 2500-dalton homopolysaccharide which we have identified as (Y 1-6 T h e phosphoenolpyruvate dependent hexose transport system is, relative to the number of macromolecules involved, a rather simple, energy-dependent, transport system. As characterized by Roseman and co-workers (Roseman, 1969) the system is composed of one soluble carrier protein, HPr, one soluble enzyme, El, and a membrane-bound complex, Ell. It catalyzes the following reactions.PEP' + HPr c -* P-HPr + pyruvate P-HPr + hexose (out) + -+ hexose-P (in) + HPr
U PVariations on this basic system have been found in Escherichia coli and other microorganisms (Saier, 1977;Postma & Roseman, 1976: Cirillo & Razin, 1973, but the elements stated above are common to all these systems. The apparent simplicity of this system makes it an attractive candidate for detailed physical-chemical studies of the transport process. Many physical techniques, however, demand large quantities of material for measurements. For this reason, we have focused our attention, initially, on developing high yield, rapid purification procedures for HPr and El (Robillard et al., 1979). During the course of the isolation, losses in HPr were encountered which were larger than expected for a single purification step, as judged by PTS activity measurements. Closer investigation has shown that these apparent losses result from the decrease in "specific activity" of HPr upon removal of an a1 -6 glucan polysaccharide. This polysaccharide functions as an effector of the PTS.' The copurification, characterization, and kinetic studies are reported below.