Abstract
We have purified a human non-pancreatic phospholipase A2 that is present in platelets and is enriched in rheumatoid synovial fluid. The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. In the E. coli phospholipase A2 assay the phospholipase exhibits an apparent specific activity of 300 mumol/mg/min. Using oligonucleotide probes based on amino-terminal sequence data, we cloned the corresponding human gene from a genomic DNA library and expressed the gene in animal cells. The protein was secreted from the cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known phospholipase A2s, and has a half-cystine pattern that is characteristic of the snake venom group II enzymes.
Highlights
Several studies have implicated PLAzs in the pathogenesis of disorders of the cardiovascular, gastrointestinal, and pulmonary systems, and skin and connective tissue [10]
The PLAz was expressed in animal cells, where it is secreted in a functionally active form
Enzyme preparations were dissolved in electrophoresis sample buffer, incubated for 10 min a t 60 "C, and subjected to SDS-PAGE on 16% gels [20].Proteins were electroblotted and stained with Coomassie Blue R-250 [21] or visualized by silver staining [20].Intransferring the PLA2 which is abnormally basic to Immobilon, we found it necessary to include 0.05% SDSin the transfer buffer
Summary
Release of Phospholipase A2 from Human Platelets-We have characterized a PLA2 from human platelets that was solubilized following the acid extraction procedure originally developed by Elsbach and co-workers [24] for PLA2 from rabbit neutrophils. The enzyme exhibits a preference for substrate presented in the physical form of E. coli membranes and hydrolyzes phospholipids presented as deoxycholate mixed micellesand sonicated liposomes at rates that aremore than 10and 100times slower, respectively. It shows a substratepreference for phosphatidylethanolamine over phosphatidylcholine. In our attempts to identify a pathogenic PLA2, we found that a predominant PLAz present in human rheumatoid synovial fluid exhibited biochemical properties with regard topH optimum, Ca2+dependence, substrate preference, and stability at acidic pHthat were identical tothe properties of the platelet PLA2. To test thispossibility we examined if acid-stable PLAz is released from platelets when they are activated under physiological conditions. The release occurred within a minute and was maximal with
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