Modified assay procedures for the phosphotransferase system in enteric bacteria.

Conditions for the assay of individual components of the bacterial phosphotransferase system (PTS) are presented wich offer two important improvements over earlier methods. First, a lactate dehydrogenase-coupled assay for phosphocarrier proteins (HPr, FPr, and Factor III^Gle) which permits their measurement in either pure or partially pure form was developed. Quantitation by this assay does not rely on the level of activity of the enzymes used. Second, conditions under which Enzyme I activity was proportional to enzyme concentration are given. With these methods levels of PTS components have been measured that are 2-to 20-fold higher than those previously reported. These levels can now account for various PTS functions measured in vivo. Further, we have shown that the phosphocarrier proteins HPr and Factor III^Gle are substrates for their respective enzymes which show typical Michaelis-Menten kineties. In addition, a method for the partial purification of Enzyme II-B^Gle essentially free of Enzyme II^Man activity is presented.