Key Points
- To determine whether aspirin inhibits prostaglandin synthase (cyclo-oxygenase) through covalent, active-site acetylation of the enzyme.
- Incubated sheep and bovine seminal vesicle microsomes and human platelet particulate fractions with radiolabeled [acetyl-3H]aspirin (200 Ci/mol).
- Resolved and detected acetylated proteins using sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).
- Evaluated competitive inhibition of acetylation using cyclo-oxygenase substrates and inhibitors, including arachidonic acid, arachidonate analogues, and indomethacin.
- Aspirin selectively acetylated a single 85,000-Da protein, which corresponded to a similarly sized acetylated protein in human platelets.
- Treatment with 100 µM aspirin resulted in 50% inhibition of prostaglandin synthase and 50% of maximal acetylation after 15 min at 37°C.
- Arachidonic acid competitively inhibited enzyme acetylation with 50% inhibition at 10–30 µM, matching its Michaelis constant as a cyclo-oxygenase substrate.
Structured PICO
PPopulationMicrosomes of sheep or bovine seminal vesicles and particulate fraction of human platelets
IInterventionAspirin (acetylsalicylic acid) and [acetyl-3H]aspirin
OOutcomeAcetylation of prostaglandin synthase (cyclo-oxygenase) and inhibition of its activitysurrogate
Aspirin acts as an active-site acetylating agent for cyclo-oxygenase, providing the biochemical mechanism for its anti-inflammatory and anti-platelet actions.