Studies on the egg-membrane lysin of tegula pfeifferi the reaction mechanism of the egg-membrane lysin

The mechanism of lysis of egg membrane was studied using pure egg-membrane lysin and the isolated egg membrane of a sea snail, Tegula pfeifferi. Kinetic analysis of the reaction and chromatographic fractionation of the reaction mixture demonstrated that the lysis can be divided into three phases in terms of lysin concentration and product release. At low concentrations of lysin (Phase I), all the lysin added was precipitated with a part of the egg membrane and nothing appeared in the supernatant. At medial concentrations of lysin (Phase II), all the lysin added precipitated and a substance(s) from egg membrane was released into the supernatant. At high concentrations of lysin (Phase III), excess lysin remained in the supernatant along with the soluble product(s) released from the egg membrane at the medial concentrations.Either when or after the lysin reacted with egg membrane, it adsorbed to an insoluble part of egg membrane and lost its activity. The maximal amount of lysin adsorbed is proportional to the amount of egg membrane. The strong bond between lysin and an insoluble part of egg membrane cannot be dissociated by 8 M urea, 0.1 M HCl, 0.1 M KOH or 10^?3 M dithiothreitol.The plot of lysin concentration versus product release showed a sigmoidal curve. In the presence of excess lysin, the amount of the product released is proportional to the amount of egg membrane. The product(s) from egg membrane is a mucopolysaccharide-protein complex(es) with a molecular weight of 5 · 10⁶ or more. Stoichiometric analysis showed that about 2000 (or more) molecules of lysin are required to liberate one molecule of the soluble product.These results strongly indicate that the lytic action of egg-membrane lysin is a stoichiometric rather than an enzymatic reaction.