Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry |
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Authors: | Yuan-Hao Hsu Darren S Dumlao Jian Cao Edward A Dennis |
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Institution: | 1. Department of Chemistry, Tunghai University, Taichung, Taiwan.; 2. Department of Pharmacology, University of California San Diego, La Jolla, California, United States of America.; 3. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.; Karolinska Institutet, Sweden, |
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Abstract: | Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and iPLA2, but not for Lp-PLA2. Our results also show that none of these PLA2s have significant PLA1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA2, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA2 assays. The sPLA2s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA2 and cPLA2, and activates sPLA2 at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA2 activity and the ability to assay the various forms of PLA2 is important in understanding its function. |
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