Conformational Changes and Activation of Phospholipase A2 Enzymes

Multiple forms of PLA2 have been isolated from almost all snake venoms but suitable analytical technique have not been available for purification and characterisation. Sequence data showed that most PLA2 isoforms did not differ in molecular weight or in net positive charge and the major differences lay in the content of negatively charged residues. We therefore concentrated on the development urea PAGE as analytical technique particularly basic urea gels. The performance of the acidic and basic urea gels has been improved to match and even exceed the resolution provided by SDS-PAGE. This has been achieved by the design of spacer gels, by the use of 8M urea and by the selection of polymerisation conditions and choice of electrolyte buffer. The basic urea gels were shown to be ideal for resolution of phospholipase A2 isoforms. Phospholipase A2 activity was recovered from both acidic and basic urea gels with high yield. The chromogenic PLA2 detection method of Shier and Trotter (1978) has been modified for use with basic urea PAGE. By using basic urea gels along and basic urea gels in chromogenic PLA2 detection method, a number of new PLA2 isoforms were identified. The basic urea-PAGE (BG) method is proposed as the basis of a simple and rapid method for the classification of PLA2 isoforms which should allow unambiguous identification of isoforms by referring bands for purified material to the isoform content of whole venoms. All phospholipase A2 enzymes tested were shown to be activated by long-chain free fatty acid and only by sub-group of acyl imidazolide. There are two major models for PLA2 activation, the first favoured by the Verger and DeHaas (1976) is the hydrophobic anchor model and second, favoured by this laboratory is the conformational change model. To distinguish between these models, we reinvestigated the phenomenon of activation of PLA2 by glutaraldehyde in the presence of long chain fatty acid. Preliminary tests showed that 15-20% 1-propanol was required for this activation. Activation was transient and continued action of glutaraldehyde progressively inactivated the enzyme but the activated state was shown to be stabilised by borohydride reduction. Enzyme activated by glutaraldehyde in the presence of 3[H]-oleic acid and stabilised with sodium borohydride lost the fatty acid on gel filtration in 20% 1-propanol but the non-radioactive enzyme remained activated. This was the first demonstration that the enzyme could be activated by conformational change. The possibility of conformational change during activation of enzyme was examined by CD and fluorescence spectroscopy. Comparison of the CD spectra of native and oleoyl imidazolide activated enzyme shows a change in secondary structure with apparent increase in both alpha-helix and beta-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of emission maximum from 341 to 348 nm. Treatment of the acidic isoform of PLA2 from the venom of Naja mossambica mossambica with oleoyl imidazolide gives a 60-fold increase in the lytic activity that can be attributed to covalent linkage of an oleoyl group to the protein. Radiolabelling experiments confirm that the bound fatty acyl chain is resistant to extraction by organic solvent or albumin during gel filtration at pH 8.0, but is rapidly eliminated under the conditions of acidic/urea , basic urea and SDS-PAGE. This indicates that the modification is stable in the absence of denaturants and must involve unstable linkage. Fluorescence and kinetic studies of activated enzyme at different pHs indicate that two groups are involve. Evidence is presented to suggest that a histidine at position 10 or 22 in sequence WWHF side-chain is the most probable site of acylation