Phospholipase from Trypanosoma brucei releases arachidonic acid by sequential sn-1, sn-2 deacylation of phospholipids.
Previously, we showed that arachidonic acid (AA) stimulates Ca2+ currents in pathogenic Trypanosoma brucei (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 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 A2 (PLA2). 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 x min(-1) x 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 PLA1 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.
Ridgley
E L
EL
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA.
Ruben
L
L
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004912:Erythrocytes / Q000469:parasitology; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D010740:Phospholipases / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D012377:Rodentia; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D001095:Arachidonic Acids; D002413:Cations, Divalent; D019216:Metals, Heavy; D010743:Phospholipids; D010740:Phospholipases