Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A(2) (PLA(2)) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA(2) (hIBPLA(2)) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA(2) (V3W-hIIAPLA(2)) and with a structurally divergent group III bee venom PLA(2) (bvPLA(2)). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp(3) of hIBPLA(2), a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA(2). Fluorescence quenching by brominated lipids indicates significant penetration of Trp(3) into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA(2) activity, suggesting that membrane insertion of hIBPLA(2) is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA(2) and bvPLA(2) by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp(3) of V3W-hIIAPLA(2), similar to that found for hIBPLA(2), and no insertion of tryptophans of bvPLA(2). Our results provide evidence that (a) structurally similar group IB and IIA PLA(2)s, but not structurally diverse group III PLA(2), significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA(2)s; and (c) structurally distinct PLA(2) isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.
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PMID:Isoform-specific membrane insertion of secretory phospholipase A2 and functional implications. 1702 99

We have monitored the composition of supported phospholipid bilayers during phospholipase A(2) hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA(2) shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5+/-3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA(2), which resides in a 21+/-1 A thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d(31)-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA(2) molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA(2).
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PMID:Distribution of reaction products in phospholipase A2 hydrolysis. 1735 73

The pH-dependent activity of phospholipase A(2) (PLA(2)) from Naja mossambica mossambica venom and the membrane-water partitioning of the lipid hydrolysis products were investigated in solid-supported palmitoyl-oleyl-phosphatidylcholine-d(31) (POPC-d(31)) membranes using neutron reflection. At pH 5, PLA(2) interacts only weakly with the substrate membrane and leads to no observable membrane breakdown, which is consistent with protonation of the catalytic histidine (His48, pK(a) approximately 6.2). The rate of the lyso-lipid partitioning into the solution phase is the same at pH 9 as at pH 7.4, and the relative membrane-water partitioning of the products is essentially the same; that is, the fatty acid accumulates in the membrane, and only the lyso-lipid is solubilized. However, Me-beta-cyclodextrin (Me-beta-CD) activates PLA(2) irrespective of pH by facilitating the solubilization of the lyso-lipid product, but not the fatty acid, of which only 22% is encapsulated at pH 9. Since no product solubilization is observed at pH 5 in the absence of Me-beta-CD, this suggests that the hydrolytic mechanism of PLA(2) is not fully disabled at pH 5 but is inhibited by a mechanism, which is counteracted by Me-beta-CD-mediated release of the lyso-lipid. Me-beta-CD does not interact with the substrate membrane, which indicates that at low pH the product extraction occurs directly from the enzyme active site outside the immediate membrane-water interface, whereas at pH 7-9, direct solubilization of the lyso-lipid from the membrane can also contribute to activation of PLA(2).
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PMID:Interfacial mechanism of phospholipase A2: pH-dependent inhibition and Me-beta-cyclodextrin activation. 1941 99