A new kinetic approach for studying phospholipase C (Clostridium perfringens .alpha. toxin) activity on phospholipid monolayers

The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phos...

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Bibliographic Details
Published in:Biochemistry (Easton) 1988-04, Vol.27 (7), p.2319-2323
Main Authors: Moreau, H, Pieroni, G, Jolivet-Reynaud, C, Alouf, J. E, Verger, R
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
AGENT PATHOGENE
alpha -toxin
Calcium - pharmacology
cations
Cholesterol
CLOSTRIDIUM
Clostridium perfringens
Clostridium perfringens - enzymology
ENZYMIC ACTIVITY
FOSFOLIPIDOS
Kinetics
lipid bilayers
Liposomes
ORGANISMOS PATOGENOS
PATHOGENS
PHOSPHATIDE
phospholipase C
PHOSPHOLIPIDS
Pressure
Surface Properties
TOXINAS
TOXINE
TOXINS
Type C Phospholipases - metabolism
Zinc - pharmacology
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Summary:The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate. It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized. No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates. By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role. The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00407a012