Synthesis of a novel acetylated neutral lipid related to platelet-activating factor by acyl-CoA:1-O-alkyl-2-acetyl-sn-glycerol acyltransferase in HL-60 cells.
Acyl-CoA:1-O-hexadecyl-2-acetyl-sn-glycerol acyl-transferase, a newly detected enzyme related to platelet-activating factor metabolism, has been characterized in microsomes of a human leukemia cell line (HL-60 cells). It has a sharp pH optimum of 6.8, does not require divalent metal ions, is stable at preincubation temperatures up to 45 degrees C, and among a variety of acyl-CoA thioesters (8:0-20:4) tested, linoleoyl-CoA is the best substrate. Km and Vmax values for 1-O-hexadecyl-2-acetyl-sn-glycerol acyltransferase are 8.5 microM and 1.7 nmol/min/mg of protein, respectively. For comparative purposes acyl-CoA:1,2-dioleoyl-sn-glycerol acyltransferase was also characterized in HL-60 microsomes. It has a relatively broad pH optimum of 6.1, is stimulated 1.4-fold by Mg2+, is relatively labile at preincubation temperatures higher than 25 degrees C, and among the various acyl-CoA thioesters tested, myristoyl-CoA is the best substrate. In substrate competition experiments, we found 1-O-hexadecyl-2-oleoyl-sn-glycerol is a competitive inhibitor (Ki = 32 microM). Our findings indicate acyl-CoA:1-O-hexadecyl-2-acetyl-sn-glycerol acyltransferase in HL-60 cells is distinctly different from acyl-CoA:1,2-dioleoyl-sn-glycerol acyltransferase. Our experimental results demonstrate that the unique enzyme activity characterized in this report also is expressed in intact HL-60 cells.
Kawasaki
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Medical and Health Sciences Division, Oak Ridge Associated Universities, Tennessee 37831.
Snyder
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D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D013696:Temperature; D014407:Tumor Cells, Cultured / Q000201:enzymology
D000214:Acyl Coenzyme A; D002413:Cations, Divalent; D010972:Platelet Activating Factor; C026562:linoleoyl-coenzyme A; D000217:Acyltransferases; C054947:acyl-CoA-1-O-alkyl-2-acetyl-sn-glycerol acyltransferase; D008274:Magnesium; D004229:Dithiothreitol