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A non-hydrolyzable phosphonate analogue of phosphatidyl inositol, racemic myo-inosityl-(1)-5-oxa-16-trifluoroacetamidohexadecyl phosphonate, was synthesized. This phosphonate inhibited the activity of phosphatidyl inositol-specific phospholipase C (PI-PLC) from Bacillus cereus with an IC50 of approximately 10 mM. Removal of the trifluoroacetyl blocking group followed by covalent binding of the phosphonate to cyanogen bromide activated Sepharose 4B via the amino group produced an affinity matrix specific for the PI-PLC from B. cereus. This affinity matrix was used to purify the phospholipase C from a complex mixture of proteins in a single step. Competition experiments with myo-inositol in the elution medium indicated that specific binding of the enzyme to the matrix most likely involves the enzyme active site. The inositol phosphonate derivatized matrix was stable over several months in neutral and alkaline media and was used repeatedly without loss of binding capacity. These results show that affinity matrices employing myo-inositol phosphonate ligands are useful for isolation and binding studies of PI-PLC and possibly of other enzymes interacting with phosphoinositides or myo-inositol phosphate derivatives.

Evidence is presented that the zymogen of porcine pancreatic phospholipase A2 (prophospholipase A2) interacts with a lipid-water interface provided that the interface has a net negative surface charge. Fluorescence spectroscopy and non-equilibrium gel filtration indicate that binding of prophospholipase A2 (proPLA) to mixed detergent micelles is dependent on the presence of an anionic detergent. Prophospholipase binding is accompanied by a change in the environment of the single tryptophan residue qualitatively similar to that observed when the active enzyme, phospholipase A2 (PLA), binds to micelles. In addition, the rate of tryptic activation of prophospholipase is significantly reduced in the presence of negatively-charged mixed micelles, whereas no change in rate occurs when neutral micelles are present. These observations suggest that the lack of catalytic activity of the zymogen toward organized substrates carrying a negative surface charge cannot be explained by a failure to bind at the lipid-water interface.