Studies on the selectivity of enzymes involved in platelet-activating factor formation in stimulated cells.
The present studies were undertaken to obtain further insight into the selectivities of the enzymes, i.e., phospholipase A2 and acetyltransferase, involved in platelet-activating factor (PAF) production upon stimulation of human polymorphonuclear leukocytes (PMN) and platelets. After appropriate stimulation of the cells in the presence of [3H]acetate the total PAF and analogs, i.e., 1-alkyl-2-acetyl-, 1-alkenyl-2-acetyl-, and 1-acyl-2-acetyl-glycero-3- phosphocholine were isolated by high performance liquid chromatography. The isolated mixture was subjected to treatment with phospholipase A1 to differentiate acetate incorporation into 1-ether linked and 1-ester linked species. The ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs amounted to 13.8 +/- 1.0 and 1.3 +/- 0.1 for PMN and platelets, respectively. When compared to the ratio of 1-ether linked and 1-ester linked species in the diradylglycerophosphocholine precursors in each cell type, i.e., 1.13 for PMN and 0.22 for platelets, these data suggested a pronounced selectivity for the phospholipase A2 and/or acetyltransferase in the process of PAF production. When the experiments were repeated with cells that had been pretreated with phenylmethanesulfonylfluoride (PMSF) to block the acetylhydrolase, the most dramatic effects were observed on acetate incorporation into 1-acyl-2-acetyl-glycero-3-phosphocholine, which increased much more than that into 1-alk(en)yl-2-acetyl-glycero-3-phosphocholine. Under these conditions, the ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs became 1.4 +/- 0.2 and 0.17 +/- 0.02 for PMN and platelets, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
van den Bosch
H
H
Centre for Biomembranes and Lipid Enzymology, University of Utrecht, The Netherlands.
Sturk
A
A
ten Cate
J W
JW
Aarsman
A J
AJ
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000085:Acetates / Q000097:blood; D000123:Acetyltransferases / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D000001:Calcimycin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007700:Kinetics; D008245:Lysophospholipase / Q000097:blood; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010741:Phospholipases A / Q000097:blood; D054466:Phospholipases A1; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000302:isolation & purification; D013917:Thrombin / Q000187:drug effects
D000085:Acetates; D010972:Platelet Activating Factor; D000001:Calcimycin; D010664:Phenylmethylsulfonyl Fluoride; D000123:Acetyltransferases; C046035:1-alkylglycerophosphocholine acetyltransferase; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D008245:Lysophospholipase; D013917:Thrombin