Identification and purification of sheep platelet phospholipase A2 isoforms. Activation by physiologic concentrations of calcium ion.
Two families of platelet phospholipase A2 activity, were chromatographically resolved by anion exchange chromatography and were functionally distinguishable by their differential phospholipid subclass substrate specificity and calcium ion requirements. The major phospholipase A2 activity was present in the cytosolic compartment, eluted from DEAE-cellulose at 230 mM NaCl (hereafter referred to as phospholipase A2(beta)), and demonstrated a 100-fold selectivity in catalyzing the hydrolysis of 1-(O)-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphocholine (plasmenylcholine) in comparisons with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (phosphatidylcholine). Phospholipase A2(beta) was purified to homogeneity by sequential gel filtration and Mono Q column chromatographies. Phospholipase A2(beta) eluted with an apparent molecular mass of 58 kDa during gel filtration chromatography and migrated as a single band with an apparent molecular mass of 30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that its native quaternary structure is dimeric. Fast protein liquid chromatography demonstrated that the polypeptides catalyzing this activity were comprised of multiple isoforms which possessed different specific activities. Each isoform required Ca2+ ion for activity and was completely activated over the range through which Ca2+ ion concentration is augmented in stimulated platelets (i.e. 300-800 nM).
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002122:Calcium Chloride / Q000494:pharmacology*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D010740:Phospholipases / Q000097:blood*; D010741:Phospholipases A / Q000097:blood* / Q000302:isolation & purification; D054467:Phospholipases A2; D012756:Sheep
D007527:Isoenzymes; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002122:Calcium Chloride