Isoelectrophoretic separation and the detection of soluble proteins containing acid-labile phosphate: use of the phosphoenolpyruvate:sugar phosphotransferase system as a model system for N1-P-histidine- and N3-P-histidine-containing proteins.
Procedures have been developed for the detection of acid-labile phosphorylations of proteins. The phosphoproteins were separated by native isoelectric focusing while maintaining the gel at about 0 degree C, and denaturing urea-Nonidet isoelectric focusing gels were adapted to run at -10 degrees C. The proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), HPr, which contains 1-P-histidine, and factor IIIglc and enzyme I, which contain 3-P-histidine when they are phosphorylated, were used to develop the conditions. Autoradiography of [32P]-labeled phosphoproteins was carried out on frozen gels which had not been acid fixed in order to avoid hydrolysis of the phosphohistidines . The frozen gels were subsequently fixed and stained, and reautoradiography revealed whether the phosphoproteins were acid stable or labile. In addition to the known proteins of the PTS, at least one other protein whose phosphorylation was dependent on enzyme I and HPr was found in Salmonella typhimurium and Escherichia coli [E.B. Waygood , and R.L. Mattoo (1983) Canad . J. Biochem. Cell Biol. 61, 150-153]. Initial experiments with rat tissues have demonstrated acid-labile phosphorylations in proteins which were either [gamma-32P]ATP or [32P]phosphoenolpyruvate dependent. The interconversion of phosphoenolpyruvate and ATP in crude extracts of bacterial cells was examined, and appropriate controls were found. Protein phosphorylation dependent upon phosphoenolpyruvate was much greater in S. typhimurium and E. coli than the corresponding ATP-dependent phosphorylation, while the opposite was found for rat tissues.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001426:Bacterial Proteins / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007525:Isoelectric Focusing / Q000379:methods*; D008099:Liver / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D011489:Protein Denaturation; D051381:Rats; D012486:Salmonella typhimurium / Q000378:metabolism
D001426:Bacterial Proteins; D010750:Phosphoproteins; D006639:Histidine; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C005436:phosphohistidine