Abstract
Previously, we showed that arachidonic acid (AA) stimulates Ca 2+ currents in pathogenic Trypanosoma brucei (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A 2 and arachidonic acid. Biochem. J 1998;336:659–666). Here we examine the mechanism used by T. brucei to release AA from the sn-2 position of diacyl glycero-phospholipids. We report that T. brucei accomplishes this feat in the apparent absence of phospholipase A 2 (PLA 2). Instead, deacylation is initiated at the sn-1 position, followed by acyl migration and hydrolysis with LysoPLA. Neither whole cell homogenates nor enriched protein fractions could release AA from substrates whose sn-1 position contained a non-hydrolyzable alkyl ether linkage. These same fractions however, released AA from ester linked phospholipids, and TLC analysis of the reaction products supported the sequential deacylation process. The release of sn-2 AA from 1-palmitoyl-2-[1- 14C]arachidonyl- sn-glycero-3-PC was linear up to 90 min at an average rate of 50 nmol min −1 mg −1. sn-2 AA was processed more efficiently than sn-2 palmitate. The reaction was also greatest for: LysoPC>diacyl-PC ( sn-1 labeled)>diacyl-PC ( sn-2 labeled). Product formation was sensitive to polar head group, and PI was processed at less than 10% the rate of PC or PE. The enzymatic deacylation was inhibited by the serine specific reagent, methyl arachidonyl fluorophosphonate (MAFP) and the cysteine reagent N-ethylmaleimide (NEM). Both NEM and MAFP inhibited LysoPLA activity under conditions where there was little effect on PLA 1 activity. Overall, we conclude that T. brucei can release AA from diacyl glycero-phospholipids by a sequential deacylation process. Two independent active sites appear to be involved. Interestingly, a high percentage of inner leaflet phospholipids are protected from degradation since they occur in the non-hydrolyzable 1-alkyl ether form.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
