Characterisation of a phospholipase C produced by Pseudomonas fluorescens.
Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures. A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield. The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis. The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography. The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine. The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed. Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents. These properties of the enzyme indicate that it is distinct from the previously reported Ps. fluorescens phospholipase C.
Ivanov
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A
Department of Biochemistry and Microbiology, University of Plovdiv, Bulgaria.
Titball
R W
RW
Kostadinova
S
S
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D008670:Metals; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D004492:Edetic Acid; D010738:Type C Phospholipases