Coexistence of two biochemically distinct phospholipase A2 activities in human platelet, monocyte, and neutrophil.
The cell-associated phospholipase A2 (PLA2) activities of the human platelet, neutrophil, and monocyte were simultaneously characterized, utilizing the biochemical differences observed between the 14 kDa (kilodalton), type II PLA2 isolated from inflammatory human synovial joint fluid (HSF) and the arachidonic acid (AA) specific, 85-kDa high molecular mass (HMM) PLA2 isolated from the cytosol of a U937 monocytic cell line. The HSF PLA2 can be distinguished from the HMM PLA2 by its resistance to acid treatment, sensitivity to a sulfhydryl reducing agent, lack of preference for the fatty acid on the sn-2 position of phospholipid substrate, and inhibition by the C-7 phosphonate-phospholipid transition-state PLA2 inhibitor. Evaluation of all three cell types revealed that HMM-like PLA2 activity was found predominantly in the cytosolic fractions, although detection in neutrophil cytosol required more concentrated preparations and the use of high specific activity [3H]AA-labeled Escherichia coli. HSP-PLA2-like activity was measured in microsomal and cytosolic fractions of all three cell types, but was found in neutrophil cytosol only after treatment with acid. Further HMM-PLA2-specific interfering agents in neutrophil cytosol were observed and exemplifies one problem in assigning the existence of this enzyme in crude broken cell preparations using activity measurements alone. The role that these two enzymes play in eicosanoid production of the respective cell types remains to be studied.
Marshall
L A
LA
Department of Inflammation and Respiratory Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939.
Roshak
A
A
D016718:Arachidonic Acid / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology* / Q000648:ultrastructure; D003600:Cytosol / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000097:blood*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009000:Monocytes / Q000201:enzymology* / Q000648:ultrastructure; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D010741:Phospholipases A / Q000097:blood* / Q000737:chemistry; D054467:Phospholipases A2; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000201:enzymology
D007527:Isoenzymes; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D004229:Dithiothreitol