The splitting of red cell membrane phospholipids by snake venom phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) was found to be promoted by either Ca2+ or a basic protein fraction from cobra venom, termed “direct lytic factor”.
In contrast, the hydrolytic attack of the venom phospholipases A on lipoprotein-bound phospholipids and pure lecithin emulsions exhibited a specific requirement for Ca2+ which could not be satisfied by direct lytic factor.
Ringhals cobra phospholipase A catalyzed the splitting of red cell ghost phospholipids in the absence of Ca2+ or direct lytic factor. Similarly, the enzymic action of phospholipase A on lecithin solubilized with the aid of sodium deoxycholate proceeded at a high rate with no activator added.
The catalytic activity of the venom phospholipases A was strongly inhibited by EDTA, regardless of whether Ca2+ of direct lytic factor served as activator. The EDTA-induced inhibition was also apparent in systems which showed no requirement for either Ca2+ or direct lytic factor. On the other hand, no inhibition resulted following pretreatment of the reaction components with EDTA and subsequent removal of the latter by dialysis. These data suggest that the inhibitory effect of EDTA is not attributable to sequestration of Ca2+ as an essential activator of the reaction system but bears directly on the catalytic function of the enzyme itself.
It is concluded that the observed disparities in the requirement of and specificity for Ca2+ or direct lytic factor of the diverse systems studied are determined both by the dissimilar molecular properties of the various phospholipases A and the differences in the physical state of the phospholipid substrates.
Some aspects concerning the mode of action of direct lytic factor are discussed.
