Electrokinetic requirements for the reaction between Cl. Perfringens α-toxin (phospholipase C) and phospholipid substrates

Abstract

1. 1. α-Toxin from Cl. perfringens (phospholipase C) has been purified until, as judged by electrophoresis and agar diffusion, a single protein component was obtained. 2. 2. The α-toxin hydrolyses lecithin in the absence of Ca 2+, provided that small amounts of a long-chain cation ( e.g. stearylamine) are present in the micelle. The rate of hydrolysis in the system is much slower than that which occurs in the presence of Ca 2+. 3. 3. The addition of the long-chain cation produced a positive electrophoretic mobility of the substrate. Complete neutralization of this net positive charge, by addition of a long-chain anion, resulted in total inhibition of enzyme activity. 4. 4. With a substrate activated with a long-chain cation, hydrolysis ceased when 60% of the lecithin molecules had been hydrolysed. Evidence is presented which indicates that this reaction is associated with reversal of the charge of the micelle from the positive to the negative state, this reversal being due to diglyceride, which eventually accumulates on the surface. 5. 5. Calcium, magnesium and uranyl salts activated the hydrolysis of lecithin emulsions and at the same time caused the micelles to become positively charged. 6. 6. Conversely, ferricyanide ions were efficient inhibitors of the enzyme and at the same time caused a reversal of the charge of the lipid micelles from positive to negative. 7. 7. A small hydrolysis of phosphatidylethanolamine by α-toxin has been demonstrated. The rate of this hydrolysis is accelerated if lecithin is added, or of it is combined as a lipoprotein. 8. 8. High-pressure ( i.e. more than 40 dynes/cm) monomolecular films of [ 32P]-lecithin are hydrolysed when Ca 2+ or UO 2 2+ are added to the bulk phase. However, the addition of long-chain cations to lecithin did not result in measurable hydrolysis of such films. Hydrolysis of low-pressure lecithin films (less than 30 dynes/cm) was observed in the complete absence of divalent cations. 9. 9. It is suggested that α-toxin can attack a phospholipid at a micelle-water interface only when the molecule is in close association with a free cationic group. When this cationic group is provided by a water-insoluble amphipathic molecule, the activation is directly related to the z-potential, i.e. net charge. On the other hand, limited hydrolysis can occur in the presence of divalent ions, e.g. Ca 2+, even when the z-potential is net negative, possibly because of localized regions of positive charges at a molecular level.