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-P-i basal salt medium with choline, as the sol e carbon and nitrogen source, was purified by precipitation with 70% s aturation ammonium sulfate in the presence of celite. 2. The PLC activ ity 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 t o purify PLC produced in a low-P-i complex medium. The resultant prepa ration was not homogeneous. 5. The molecular weight for both PLC prepa rations was about 70 kDa. 6. Both PLC used phosphatydilcholine and sph ingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K-M of 25 mM for p-nitrophenylphosphorylcholine. 7. They wer e not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100. 8. 2% sodium dodecylsulfate and 1% tetradecyltrimethyla mmonium bromide inhibited the PLC from the HP1-BSM plus choline but no t the enzyme from the LP(1)-CM.