A Phospholipase C from the Dallas 1E Strain of Legionella pneumophila Serogroup 5: Purification and Characterization of Conditions for Optimal Activity with an Artificial Substrate
Phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5 was purified from buffered yeast extract culture supernate by ion-exchange chromatography followed by fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme activity was assayed by hydrolysis of p-nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritiated L-α-dipalmitoylphosphatidylcholine labelled in the methyl groups of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassie-blue staining. This protein migrated with an apparent Mr of 50000–54000. Phospholipase C activity was maximal at pH ≥ 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS. EDTA, Cu2+, Fe2+ and Zn2+ inhibited enzyme activity, whereas Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme.
BaineW.B.,
RasheedJ.K.1979; Aromatic substrate specificity of browning by cultures of the Legionnaires’ disease bacterium. Annals of Internal Medicine 90:619–620
BaineW.B.,
RasheedJ.K.,
FeeleyJ.C.,
GormanG.W.,
CasidaL.E.Jr1978; Effect of supplemental l-tyrosine on pigment production in cultures of the Legionnaires’ disease bacterium. Current Microbiology 1:93–94
BerkaR.M.,
VasilM.L.1982; Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa:purification and preliminary characterization. Journal of Bacteriology 152:239–245
De VriesT.1961; Potentials of electrochemical reactions at 25 °C. In Handbook of Chemistry and Physics, 43rd edn.. pp. 1743–1745 Edited by
HodgmanC. D.,
WeastR. C.,
ShanklandR. S.,
SelbyS. M.
Cleveland: The Chemical Rubber Publishing Co.;
El-SayedM.Y.,
RobertsM.F.1985; Charged detergents enhance the activity of phospholipase C (Bacillus cereus) towards micellar short-chain phosphatidylcholine. Biochimica et biophysica acta 831:133–141
El-SayedM.Y.,
De BoseC.D.,
CouryL.A.,
RobertsM.F.1985; Sensitivity of phospholipase C (Bacillus cereus) activity to phosphatidylcholine structural modifications. Biochimica et biophysica acta 837:325–335
GreenA.A.,
HughesW.L.1955; Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents. Methods in Enzymology 1:67–90
GrossmanS.,
OestreicherG.,
HogueP.K.,
CobleyJ.G.,
SingerT.P.1974; Microanalytical determination of the activities of phospholipases A, C, and D and of their mixtures. Analytical Biochemistry 58:301–309
GuligP.A.,
HansenE.J.1985; Coprecipitation of lipopolysaccharide and the 39000-molecular-weight major outer membrane protein of Haemophilus influenzae type b by lipopolysaccharide-directed monoclonal antibody. Infection and Immunity 49:819–827
HamesB.D.1981; An introduction to polyacrylamide gel electrophoresis. In Gel Electrophoresis of Proteins pp. 1–91HamesB.D.,
RickwoodD.
Edited by Oxford: IRL Press;
HindahlM.S.,
IglewskiB.H.1986; Outer membrane proteins from Legionella pneumophila serogroups and other Legionella species. Infection and Immunity 51:94–101
HoffmanP.1984; Bacterial physiology. In Legionella, Proceedings of the 2nd International Symposium, pp. 61–67ThomsberryC.,
BalowsA.,
FeeleyJ. C.,
JakubowskiW.
Edited by Washington, DC: American Society for Microbiology;
HorwitzM.A.1983; The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. Journal of Experimental Medicine 158:2108–2126
KlausM.H.,
ClementsJ.A.,
HavelR.J.1961; Composition of surface-active material isolated from beef lung. Proceedings of the National Academy of Sciences of the United States of America 47:1858–1859
KuriokaS.,
MatsudaM.1976; Phospholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme. Analytical Biochemistry 75:281–289
MalmqvistT.,
ThelestamM.,
MöllbyR.1984; Hydrophobicity of cultured mammalian cells and some effects of bacterial phospholipases C. Acta pathologica, microbiologica et immunologica scandinavica B92:127–133
MöllbyR.1978; Bacterial phospholipases. In Bacterial Toxins and Cell Membranes, pp. 367–424JeljaszewiczJ.,
WadströmT.
Edited by New York: Academic Press;
OtaniS.,
MatsuiI.,
KuramotoA.,
MorisawaS.1984; Induction of ornithine decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism. Biochimica et biophysica acta 800:96–101
PineL.,
GeorgeJ.R.,
ReevesM.W.,
HarrellW.K.1979; Development of a chemically defined liquid medium for growth of Legionella pneumophila.
. Journal of Clinical Microbiology 9:615–626
TurnerJ.C.,
AndersonH.M.,
GandalC.P.1958; Species differences in red blood cell phospha-tides separated by column and paper chromatography. Biochimica et biophysica acta 30:130–134
A Phospholipase C from the Dallas 1E Strain of Legionella pneumophila Serogroup 5: Purification and Characterization of Conditions for Optimal Activity with an Artificial Substrate