Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

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 A 2 (PLA 2 ) in vivo . We have developed mass spectrometric methodology for analyzing PLA 2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA 2 , cPLA 2 and iPLA 2 , but not for Lp-PLA 2 . Our results also show that none of these PLA 2 s have significant PLA 1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA 2 , we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA 2 assays. The sPLA 2 s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA 2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA 2 and cPLA 2 , and activates sPLA 2 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 PLA 2 activity and the ability to assay the various forms of PLA 2 is important in understanding its function.