Phospholipase D and phosphatidic acid enhance the hydrolysis of phospholipids in vesicles and in cell membranes by human secreted phospholipase A2.

Phosphatidyl-choline (PC) vesicles and normal cell membranes are resistant to hydrolysis by human group II secreted PLA2, an enzyme that can attain high concentrations in extracellular fluids during many inflammatory processes. This highly cationic enzyme (pI>10.5) has a marked preference for anionic phospholipid interfaces, normally present within the cell. Therefore, the ability of one such anionic phospholipid, phosphatidic acid (PA), to enhance the activity of this enzyme has been investigated in detail. Results using model membrane vesicles and a continuous fluorescence assay highlight the ability of low molar proportions of PA to stimulate vesicle hydrolysis and this stimulation with increasing PA was parallelled by enhanced interfacial binding. In contrast, no productive binding of this enzyme could be detected to the surface of pure PC vesicles. The enhancement of hydrolysis in the presence of PA could also be achieved by prior treatment of pure PC vesicles with PLD, an effect that was dependent on the concentration of PLD and the duration of exposure to this enzyme. The fluorescence assay also allowed cell membranes and whole cells to be used as substrates and whereas such membrane presentations were refractory to hydrolysis by the human enzyme, prior treatment with PLD allowed hydrolysis using concentrations of this PLA2 that would be found extracellularly under inflammatory conditions. These results highlight the potential for PA, generated at the surface of the cell membrane, to be hydrolysed by extracellular human sPLA2 with the generation of lysophosphatidic acid and other lipid mediators and provides one possible mechanism whereby this human sPLA2 could become pro-inflammatory.

Cardiolipin hydrolysis by human phospholipases A2. The multiple enzymatic activities of human cytosolic phospholipase A2.

The ability of mammalian phospholipases A2 (PLA2) to hydrolyse cardiolipin (diphosphatidylglycerol) was monitored with a fluorescent displacement assay which allows the use of natural phospholipid substrates. The mammalian enzymes used were porcine pancreatic (Group I) secretory PLA2 (sPLA2), human non-pancreatic (Group II) sPLA2 and human cytosolic PLA2 (cPLA2). High activity was observed with porcine pancreas sPLA2 whereas the human sPLA2 demonstrated only minimal activity with this substrate. In comparison, sPLA2 from Naja naja venom (Group I) also showed only modest activity with this substrate. Since many lipases possess PLA1 activity, a representative enzyme from Rhizopus arrhizus was also assessed for its ability to hydrolyse cardiolipin which proved to be a good substrate for this fungal lipase. In all cases dilysocardiolipin was the major product while some monolyso intermediate was detected after chromatographic separation. Human cPLA2 was unable to hydrolyse cardiolipin at a significant rate, however, both monolysocardiolipin and dilysocardiolipin, which are prepared by the PLA2-catalysed hydrolysis of cardiolipin, were good substrates providing a further example of the extensive lysophospholipase activity of this enzyme. Moreover, cardiolipin that was initially hydrolysed in situ with either excess porcine pancreatic PLA2 or R. arrhizus lipase (PLA1) was subsequently hydrolysed by human cPLA2. One explanation of this result is that human cPLA2 is able to hydrolyse both 1-acyl and 2-acyl-lysophospholipids. (c) 1998 Elsevier Science B.V.

The hydrolysis of phosphatidyl-alcohols by phospholipases A2: effect of head group size and polarity.

The ability of a variety of secretory phospholipases A2 (sPLA2: EC 3.1.1.4) to bind to and hydrolyse a series of phosphatidyl-alcohol substrates, in the absence of detergent, was explored by both fluorescence-based kinetic and interfacial binding assays. The enzymes used were sPLA2 from porcine pancreas, Naja naja venom and a recombinant human non-pancreatic enzyme. Four dioleoyl phosphatidyl-alcohols were used with different headgroups, methanol, ethanol, propanol and butanol. Comparative kinetic analyses with dioleoyl phosphatidyl-choline, dioleoyl phosphatidyl-glycerol and wheat germ phosphatidyl-inositol are also described. With the phosphatidyl-alcohol series, as the headgroup acyl-chain length increased the susceptibility to hydrolysis decreased. This effect was much more pronounced with the human non-pancreatic and the Naja naja venom enzymes than with the pancreatic enzyme. Maximum activity in this assay system was observed with porcine pancreatic sPLA2 and dioleoyl phosphatidyl-methanol (1440 +/- 167 micromol/min/mg). We demonstrate that the slow rate of hydrolysis of dioleoyl phosphatidyl-propanol by the human non-pancreatic secretory enzyme (4.56 +/- 0.90 micromol/min/mg) is not due to a lack of interfacial binding. The hydrolysis of mixtures of dioleoyl phosphatidyl-choline and dioleoyl phosphatidyl-propanol in various molar proportions by Naja naja sPLA2 suggests good mixing of the two phospholipids with minimal phospholipid domain formation under these assay conditions. We present strong evidence for a stimulation of hydrolysis of phosphatidyl-choline by human non-pancreatic sPLA2 in the presence of as little as 1 mol% phosphatidyl-methanol (<40 fold total rate enhancement). Overall, the results demonstrate that the rates of hydrolysis of anionic phospholipids by sPLA2 vary considerably with the different enzymes from this close structurally related family. The tight binding of the human enzyme to poorly hydrolysable anionic phospholipid vesicles provides a novel mechanism of enzyme inhibition by interfacial sequestration.

Enhanced hydrolysis of phosphatidylcholine by human group II non-pancreatic secreted phospholipase A2 as a result of interfacial activation by specific anions. Potential role of cholesterol sulphate.

The extracellular concentration of the Group II human non-pancreatic secreted phospholipase A2 (hnpsPLA2) is elevated in a variety of inflammatory disorders. This enzyme is remarkable because it demonstrates almost zero activity with egg phosphatidylcholine (PC) or synthetic dioleoyl-phosphatidylcholine (DOPC) as substrate, but expresses high activity with the anionic phospholipid dioleoyl-phosphatidylglycerol (DOPG), a feature shared with the Group II enzyme from rat liver. The presence of certain membrane-bound anions can enhance hydrolysis of PC by the mammalian secreted PLA2S. In this study the ability of various non-polar anions to stimulate DOPC hydrolysis by secreted PLA2S has been investigated. The naturally occurring membrane anion, cholesterol sulphate, was particularly effective in stimulating the hydrolysis of both DOPC and also 1-stearoyl-2-arachidonyl phosphatidylcholine by hnpsPLA2. Activation of DOPC hydrolysis was also achieved with dioleoyl-phosphatidylserine (DOPS); however, DOPS was less effective than cholesterol sulphate. In contrast, the dianion dioleoyl-phosphatidic acid, a known activator of pig pancreatic PLA2, failed to activate the human enzyme. It remains to be established whether cell plasma-membrane hydrolysis by extracellular hnpsPLA2 can be activated in vivo by the presence of suitable membrane anions such as cholesterol sulphate and thus promote an inflammatory response within the cell.

A continuous fluorescence displacement assay for phospholipase A2 using albumin and medium chain phospholipid substrates.

A continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino) undecanoic acid from albumin by decanoic acid released as a result of phospholipase A2-catalyzed hydrolysis of didecanoyl-phosphatidylcholine. Using the phospholipase A2 from Naja naja, this assay will detect activity from 1 ng of pure enzyme and a linear response in terms of fluorescence change with time is observed up to about 50 ng of enzyme. The assay is compared with the original fluorescence displacement assay (Wilton, D.C., 1990, Biochem. J. 266, 435-439) which uses rat liver fatty acid binding protein instead of albumin and is able to utilize any natural long chain phospholipid as substrate. The present assay will provide a very convenient method for detection during enzyme purification of the low molecular weight secreted phospholipase A2 and other phospholipases A that can hydrolyze medium chain phospholipids. The assay should also allow the identification of inhibitors of these enzymes. Other substrates including dioleoylphosphatidylcholine and 1-stearoyl-2-arachidonoylphosphatidylcholine were also evaluated in the assay.