Assessing Phospholipase A(2) 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.