Purification and characterization of a 65-kilodalton diisopropylfluorophosphate-binding protein in the outer membrane of Fusobacterium nucleatum Fev1.
A 65-kilodalton protein was identified in the outer membrane of Fusobacterium nucleatum Fev1 by SDS-PAGE. The relative amount of the protein varied during growth, being greatest in stationary phase. The protein was radio-labeled by [125I]-lactoperoxidase treatment of live cells and was only partially extractable with 2% sodium dodecylsulfate (SDS) at room temperature, and therefore assumed to be both exposed to the cell surface and peptidoglycan associated. In intact cells the protein bound diisopropylfluorophosphate (DFP), indicating that it may be a serine protease. DFP-binding activity depended apparently on proper localization of the proteins in the membrane. Several methods were tried in attempts to purify the 65-kilodalton protein; the method that gave best results was preparative SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to about pH 5. Amino acid composition analysis showed that the 65-kilodalton protein possesses an excess of hydrophilic over hydrophobic residues, polarity index 52%. The N-terminal end of the protein was apparently blocked.
Brokstad
K A
KA
Department of Biochemistry, University of Bergen, Norway.
Jensen
H B
HB
D000596:Amino Acids / Q000032:analysis; D001345:Autoradiography; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005673:Fusobacterium / Q000032:analysis*; D007457:Iodine Radioisotopes; D007525:Isoelectric Focusing; D007531:Isoflurophate / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D011485:Protein Binding; D012967:Sodium Dodecyl Sulfate; D014316:Tritium
D000596:Amino Acids; D001426:Bacterial Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D014316:Tritium; D007531:Isoflurophate; D012967:Sodium Dodecyl Sulfate; D010450:Endopeptidases