Biosynthesis of paf-acether. XI. Regulation of acetyltransferase by enzyme-substrate imbalance.
Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.
Ninio
E
E
INSERM U.200, Université Paris-Sud, Clamart, France.
Joly
F
F
Bessou
G
G
D000123:Acetyltransferases / Q000097:blood* / Q000302:isolation & purification; D006706:Homeostasis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology*; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D010972:Platelet Activating Factor; D012710:Serum Albumin, Bovine; D000123:Acetyltransferases; C052591:alkylglycerophosphate 2-O-acetyltransferase