Reassembly of a fimbrial hemagglutinin from Pseudomonas solanacearum after purification of the subunit by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Distilled water homogenates of Pseudomonas solanacearum B1, a highly fimbriated strain, strongly agglutinated human group A erythrocytes. The fimbriae and hemagglutinating activity were precipitated from the crude extract with 1% acetic acid, redissolved at pH 10, and precipitated again with 20 mM CaCl2 at pH 6.9. Ca2+, Mg2+, and Zn2+ had similar ability to precipitate the fimbrial hemagglutinin, but Na+ and K+ were much less effective. The fimbrial protein in the precipitate was purified to homogeneity by preparative gel electrophoresis in sodium dodecyl sulfate. The major protein band was eluted, and sodium dodecyl sulfate was removed by chromatography on ion retardation resin (AG 11A8) in 6 M urea. After dialysis against 10 mM sodium acetate (pH 4.5) to remove the urea, the protein reassembled to yield long fibers. These fibers were identical to fimbriae in the crude extract in diameter (6 nm) and in their ability to cause hemagglutination. The purified fimbriae contained no carbohydrates and wee similar to other bacterial fimbriae in amino acid composition, with hydrophobic amino acids comprising 41.8% of the total.
Young
D H
DH
Stemmer
W P
WP
Sequeira
L
L
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis; D001789:Blood Group Antigens; D002241:Carbohydrates / Q000032:analysis; D002412:Cations / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010861:Fimbriae, Bacterial / Q000276:immunology*; D006384:Hemagglutination; D006388:Hemagglutinins / Q000032:analysis / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D011549:Pseudomonas / Q000276:immunology* / Q000648:ultrastructure
D000596:Amino Acids; D001426:Bacterial Proteins; D001789:Blood Group Antigens; D002241:Carbohydrates; D002412:Cations; D006388:Hemagglutinins