A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline.
1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-Pi basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite. 2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate. 3. The peak containing the PLC activity revealed a single protein after SDS-PAGE. 4. The method could also be applied to purify PLC produced in a low-Pi complex medium. The resultant preparation was not homogeneous. 5. The molecular weight for both PLC preparations was about 70 kDa. 6. Both PLC used phosphatidylcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent KM of 25 mM for p-nitrophenylphosphorylcholine. 7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100. 8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP1-BSM plus choline but not the enzyme from the LP1-CM.
Lucchesi
G I
GI
Departmento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Córdoba, Argentina.
Domenech
C E
CE
D002794:Choline / Q000494:pharmacology; D003470:Culture Media; D010710:Phosphates / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015203:Reproducibility of Results; D010738:Type C Phospholipases / Q000187:drug effects / Q000302:isolation & purification*
D003470:Culture Media; D010710:Phosphates; D010738:Type C Phospholipases; D002794:Choline