title: Assessing phospholipase A2 activity toward cardiolipin by mass spectrometry.
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.
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan. howardhsu@thu.edu.tw
authors.forename: Yuan-Hao
authors.initials: YH
authors.lastname: Hsu
authors.forename: Darren S
authors.initials: DS
authors.lastname: Dumlao
authors.forename: Jian
authors.initials: J
authors.lastname: Cao
authors.forename: Edward A
authors.initials: EA
authors.lastname: Dennis
mesh_terms: D000818:Animals; D002308:Cardiolipins / Q000378:metabolism*; D054513:Group IV Phospholipases A2 / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D054467:Phospholipases A2 / Q000378:metabolism*; D054512:Phospholipases A2, Calcium-Independent / Q000378:metabolism; D054497:Phospholipases A2, Secretory / Q000378:metabolism
chemical_list: D002308:Cardiolipins; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D054512:Phospholipases A2, Calcium-Independent; D054497:Phospholipases A2, Secretory