Comparison of the properties of human group II phospholipase A2 with other secretory phospholipases.

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The human group II secretory phospholipase A, (sPLA,) has been implicated in a number of inflammatory conditions, including rheumatoid arthritis. A continuous fluorescence displacement assay which measures the release of long chain fatty acids [l] has been used to investigate the activity of a recombinant form of this human enzyme (HR PLAJ and is reported here for the first time. Secretory phospholipases 4 act by interfacial catalysis. Their activity is dependent on phospholipid aggregates presenting a suitable lipidhater interface. Enzymes from different sources express different activities depending on the characteristics of the interface. The interfacial characteristics depend, to a large extent, on the headgroup of the phospholipid substrate. By comparing the rates of hydrolysis achieved using substrates with different headgroups information concerning the preferred interface characteristics can be acquired. In this study we have assayed the activity of PLA,'s using dioleoylphosphatidylcholine (DOPC). dioleoyl-phosphatidylethanolamine (DOPE) and dioleoyl-phosphatidylglycerol (DOPG) as substrates. These results have been compared with others [2] for sPLA,'s from Naia naia (NN), Crotalus atrox (CA) and bee (BV) venoms and the mammalian enzymes from porcine pancreas (PP) and rat liver (RL) [3]. Whole cells have also been used as a source of substrate phospholipids. Assays were performed according to the methods previously described [2]. Phospholipids were presented by direct injection of 50 pl of a 10 mg/ml solution of substrate in methanol to 10 ml of assay buffer (100 mM Tris-HCI, 100 mM NaCI, 5 mM CaCI,, pH 8.0). Assays using the recombinant human and rat liver derived enzymes were performed at 37 C. For hydrolyses using whole cells as substrate, approx. 50 000 hepatocyte cells were taken in 1 ml of Hanks balanced salt solution (free of albumin). DAUDA (1 nmole) and FABP (1 nmole) were added as for the normal assay and hydrolysis started by addition of the appropriate s P 4 . The fluorescence displacement traces for hydrolyses of DOPC, DOPE and DOPG by HR PLA, are shown in Figure 1. These results demonstrate that HR PLA, has a dramatic preference for the negatively charged DOPG as compared to DOPC. Indeed. the rate of hydrolysis observed using phosphatidylcholine as substrate is virtually zero. DOPE is hydrolysed at a faster rate than DOPC. but much slower than DOPG. The rates relative to DOPC are DOPC (1.00 f 0.24). DOPE (4.68 f 0.21) and DOPG (383 f 42).