Inhibition of secreted phospholipases A2 by annexin V.: Competition for anionic phospholipid interfaces allows an assessment of the relative interfacial affinities of secreted phospholipases A2

The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A(2) (PLA(2)) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. In this investigation we have examined the substrate depletion mechanism of annexin V using a variety of phospholipid substrates and secreted PLA(2)'s (sPLA(2)). The results suggest that the term interfacial competition best describes the inhibitory effect of annexin V although the overall inhibitory process remains one of substrate sequestration by the annexin. We have utilised the competitive nature of the interaction of enzyme and annexin V for a phospholipid interface as a means of quantifying the relative affinity of sPLA(2)'s for anionic phospholipid vesicles. The results highlight the very high affinity of the human non-pancreatic sPLA(2) for such vesicles (K-d << 10(-10) M) while the Naja naja venom PLA(2) and porcine pancreatic sPLA(2) showed lower affinities. Hydrolysis of mixed vesicles containing phosphatidylserine and phosphatidylcholine by the venom and pancreatic enzymes were differentially inhibited by annexin V. This difference must reflect the preference of both annexin V and the pancreatic enzyme for an anionic phospholipid interface. In contrast, the venom enzyme is able to readily hydrolyse phosphatidylcholine domains that would be minimally affected by annexin V. Annexin V was an effective inhibitor of cardiolipin hydrolysis by the pancreatic PLA(2), however the inhibition was of a more complex nature than seen with other phospholipids tested. Overall the results highlight the ability of annexin V to inhibit phospholipid hydrolysis by sPLA(2)'s by an interfacial competition (substrate depletion) mechanism. The effectiveness of annexin V as an apparent inhibitor depends on the nature of the enzyme and the phospholipid substrate. (C) 1998 Elsevier Science B.V.