Purification of two Bacillus subtilis proteins which cross-react with antibodies directed against eukaryotic protein kinase C, the His HPr kinase and trigger factor.
As in eukaryotes, phosphorylation of Ser residues in proteins appears to be common phenomenon in bacteria. Surprisingly, however, very few Ser/Thr protein kinases have been identified and in this study antibodies directed against mammalian protein kinase C (PKC) have been used in attempts to isolate conserved Ser/Thr protein kinases. Using the mAb M7 against rat brain PKC, a single 70 kDa band was identified in total cell extracts of Bacillus subtilis by Western blotting after SDS-PAGE, whilst using polyclonal antibody alpha-PKC1p against Saccharomyces cerevisiae PKC a single 67 kDa band was identified by the same procedure. The two proteins were purified independently on the basis of antibody recognition employing two-dimensional gel electrophoresis as a final step, which allowed subsequent microsequencing. The 70 kDa band was thus identified as the phosphoenolpyruvate-dependent His HPr kinase, Enzyme 1 of the phosphotransferase system. This identity was confirmed using a mutant deleted for ptsl, encoding Enzyme 1. The 67 kDa protein was identified as a previously unknown B. subtilis 'trigger factor', homologous to an Escherichia coli protein-folding enzyme, peptidylprolyl cis-trans-isomerase implicated in cell division.
Zouari
Naïla
N
Institut de Génétique et Microbiologic, URA 2225, University Paris XI, Bâtiment 409, 91405 Orsay cedex, France.
Roche
Benoît
B
Institut de Génétique et Microbiologic, URA 2225, University Paris XI, Bâtiment 409, 91405 Orsay cedex, France.
Seegers
Jos F M L
JFML
Institut de Génétique et Microbiologic, URA 2225, University Paris XI, Bâtiment 409, 91405 Orsay cedex, France.
Séror
Simone J
SJ
Institut de Génétique et Microbiologic, URA 2225, University Paris XI, Bâtiment 409, 91405 Orsay cedex, France.
D000591:Amino Acid Isomerases / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001921:Brain / Q000201:enzymology; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D003429:Cross Reactions; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics* / Q000276:immunology; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000235:genetics* / Q000276:immunology; D011493:Protein Kinase C / Q000235:genetics / Q000276:immunology; D011494:Protein Kinases / Q000235:genetics* / Q000276:immunology; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D002352:Carrier Proteins; D011494:Protein Kinases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D011493:Protein Kinase C; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase