title: Purification and some properties of phospholipase C from Achromobacter xylosoxidans.
abstract: A non-haemolytic phospholipase C (EC 3.1.4.3) was purified from the culture medium of Achromobacter xylosoxidans with a 5% yield and a purification factor of 330. A combination of ultrafiltration, acetone precipitation and two subsequent affinity chromatographic steps was used. The affinity chromatography is a new application of 2-(4-aminophenylsulphonyl)ethyl-cellulose, a sorbent that has previously been used for the purification of phospholipase C from Bacillus cereus. The purified enzyme gave four distinct bands on polyacrylamide gel electrophoresis, and each band was catalytically active. Under our experimental conditions, the phospholipids examined were hydrolysed in the following order: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin. Neither the synthetic substrate p-nitrophenylphosphorylcholine nor phosphatidylinositol was hydrolysed under different experimental conditions. For maximal hydrolytic activity toward phosphatidylcholine, the enzyme required Triton X-100 and Ca2+ ions. EDTA was inhibitory, but the enzyme activity was almost completely restored by Zn2+. The molecular mass of the phospholipase C, estimated by gel permeation, was 34,000 daltons.
authors.affiliation: Department of Biochemistry and Microbiology, University of Plovdiv, Bulgaria.
authors.forename: S
authors.initials: S
authors.lastname: Kostadinova
authors.forename: A
authors.initials: A
authors.lastname: Ivanov
authors.forename: E
authors.initials: E
authors.lastname: Kamberov
mesh_terms: D000421:Alcaligenes / Q000201:enzymology*; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D003470:Culture Media / Q000737:chemistry; D003902:Detergents; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D013696:Temperature; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism; D014462:Ultrafiltration
chemical_list: D002413:Cations, Divalent; D003470:Culture Media; D003902:Detergents; D004492:Edetic Acid; D010738:Type C Phospholipases