The trypsin-catalyzed hydrolysis of monomolecular films of lysylphosphatidylglycerol

The hydrolysis by trypsin of the bacterial phospholipid, lysylphosphatidyl-glycerol has been studied at the air-water interface. High specific activity [14C]-lysylphosphatidylglycerol was prepared biosynthetically and the trypsin action followed by measuring the loss of surface radioactivity from a monolayer of the phospholipid spread on a water surface. After a lag phase, hydrolysis proceeded at a constant rate proportional to the enzyme concentration until 50–85% of the substrate had been hydrolysed. Maximum hydrolysis occurred between 10–35 dynes/cm surface pressure and at pH 7.5–8.5. A slight fall in the surface pressure occurred but this was less than predicted from the force-area curves of lysylphosphatidylglycerol and phosphatidylglycerol. Ca2+ (0.5 mM) and to a lesser extent Mg2+ and Sr2+ stimulated the reaction. During the reaction 45Ca2+ became bound on the monolayer in proportion to the extent of the enzymic hydrolysis. The stimulation by calcium required the synergic presence of low concentrations of Tris buffer. High concentrations of CaCl2 (2 mM), NaCl (25 mM), Tris buffer (47 mM) almost completely inhibited the reaction. Chlorpromazine, which removes Ca2+ from phospholipid membranes, markedly stimulated hydrolysis at 2.5–6.0 μM, but only in the presence of added calcium: higher concentrations of chlorpromazine (0.125 mM) completely inhibited the reaction