Enzymatic hydrolysis of immobilized sphingomyelin by three bacterial phospholipases C.
Through hydrophobic interaction, sphingomyelin was adsorbed to agarose beads containing octyl groups by a stepwise dilution procedure. This immobilized lipid was used as a substrate for three bacterial phospholipases C (E.C. 3.1.4.3.). The degradation with time of this substrate showed two different fractions of the substrate according to hydrolysing velocity in the early part of the time-curve when phospholipases C from Bacillus cereus and Clostridium perfringens were used. The early fractions could be predigested by the enzymes, a procedure which resulted in linear time-curves. The corresponding early part of the time-curve for phospholipase C from Staphylococcus aureus was linear, indicating a comparatively large early fraction of the substrate for this enzyme. The stock gel of the immobilized lipid substrate could be stored for months. It was easily and reproducibly handled as a water suspension. After enzymatic hydrolysis the substrate was rapidly separated from enzyme and product by filtration. The enzyme assay presented thus represents a convenient way to avoid the difficulties connected with the use of temporary sonicated suspensions as substrate for bacterial phospholipases C.
D001409:Bacillus cereus / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D003016:Clostridium perfringens / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008722:Methods; D010740:Phospholipases / Q000378:metabolism*; D010767:Phosphorylcholine / Q000494:pharmacology; D013109:Sphingomyelins / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology*; D010738:Type C Phospholipases / Q000378:metabolism*
D002413:Cations, Divalent; D013109:Sphingomyelins; D010767:Phosphorylcholine; D010740:Phospholipases; D010738:Type C Phospholipases