A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline |
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| Institution: | 1. Queen''s University Belfast, School of Biological Sciences, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom;2. Department of Physiology, Cornell University Weill Medical College, New York, NY 10065, USA;1. Department of Cell and Molecular Biology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia;2. Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia;3. Institute of Bioscience Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia;1. Laboratory of Toxicology, Medical School, University of Crete, Voutes, 71409 Heraklion, Crete, Greece;2. Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Beyazit, Istanbul 34116, Turkey;3. Laboratory of Anatomy, Medical School, University of Crete, Voutes, 71110 Heraklion, Crete, Greece;4. Department of Biochemistry and Biotechnology, University of Thessaly, Ploutonos 26 & Aiolou, 41221 Larissa, Greece;5. General Chemical State Laboratory of Greece, Department of Hazardous Substances, Mixtures and Articles, 16 An. Tsocha, 1152 Athens, Greece;6. UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal |
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| Abstract: | - 1.1. 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.
- 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
- 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
- 4.4. The method could also be applied to purify PLC produced in a low-Pi complex medium. The resultant preparation was not homogeneous.
- 5.5. The molecular weight for both PLC preparations was about 70 kDa.
- 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent KM of 25 mM for p-nitrophenylphosphorylcholine.
- 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
- 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HPl-BSM plus choline but not the enzyme from the LPl-CM.
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