Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388.
Exoenzyme S was purified > 1,500-fold from the culture supernatant fluid of Pseudomonas aeruginosa 388 at high yield without utilization of solvents or detergents. Two proteins, with apparent molecular sizes of 53 and 49 kDa, cofractionated with exoenzyme S activity. Rabbit anti-49-kDa-protein immunoglobulin G was prepared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified 49-kDa protein as immunogen. Anti-49-kDa-protein IgG inhibited the ADP-ribosyltransferase activity of purified exoenzyme S in a dose-dependent manner, which indicated a role for the 49-kDa protein in the ADP-ribosylation reaction. Analysis by ultrafiltration showed that exoenzyme S activity and the 53- and 49-kDa proteins cofractionated and that exoenzyme S was apparently > 300 kDa in size. Urea (8 M) and 1.0% Triton X-100 reversibly decreased the apparent molecular sizes of exoenzyme S activity and the 53- and 49-kDa proteins to between 30 and 100 kDa.
Kulich
S M
SM
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Frank
D W
DW
Barbieri
J T
JT
D036002:ADP Ribose Transferases; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000918:Antibody Specificity; D001427:Bacterial Toxins; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000494:pharmacology; D051379:Mice; D017830:Octoxynol; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D014462:Ultrafiltration; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D001427:Bacterial Toxins; D007074:Immunoglobulin G; D011092:Polyethylene Glycols; D014508:Urea; D017830:Octoxynol; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S