Identification and characterization of a new enzyme of the group “phospholipase D” isolated from Corynebacterium ovis

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1. 1. A new enzyme belonging to the group of phospholipases D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was found in Corynebacterium pseudotuberculosis (ovis). It splits sphingomyelins to N- acylsphingosylphosphates and choline, and lysophosphatidylcholine to lysophosphatidic acids and choline. 2. 2. Methods of purification of the enzyme by (NH 4) 2SO 4 and methanol precipitation and elution from CM Sephadex columns are described. 3. 3. The behaviour of the enzyme upon Sephadex columns, ion-exchangers and electrophoresis was investigated. 4. 4. Optimal conditions of the enzymic reaction are studied and the K m determined using sphingomyelins as a substrate (K m = 6.2 mM) and lysophosphatidylcholines ( K m = 8.3 mM) . 5. 5. Using a new modification of paper and thin-layer chromatography the product of enzymic hydrolysis of sphingomyelins, N- acylsphingosylphosphates , were separated from other related compounds. 6. 6. A new colour reaction for the characterization of N- acylsphingosylphosphates is described. 7. 7. The spectrum of sphingosine bases in sphingomyelins and N- acylsphingosylphosphates is characterized by 4-sphingenine (as the main component) and sphinganine (present only in a trace amount). 8. 8. The main fatty acids are in both sphingolipids C 24:1 and C 16:0 acids. The minor components are composed from C 24:0, C 22:0, C 18:0, C 18:1, C 22:1, C 23:0 and C 20:0 acids.