Biochemical and Biophysical Research Communications
Selective oxygenation of N-arachidonylglycine by cyclooxygenase-2☆
Section snippets
Materials and methods
Chemicals. AA was obtained from NuChek Prep (Elysian, MN). N-Arachidonylglycine was purchased from Cayman Chemical (Ann Arbor, MI). Ram seminal vesicles were obtained from Oxford Biomedical Research (Oxford, MI). Hematin and triphenylphosphine were purchased from Sigma. All other chemicals were from Aldrich.
Enzymology. COX-1 was purified from ram seminal vesicles as previously described [19]. Site-directed mutagenesis of COX-2 was performed as described previously [20]. Recombinant COX-2
Oxygenation of NAGly by purified COX-2
Incubation of NAGly with purified recombinant murine COX-2 (mCOX-2) gave 40% of the initial rate of O2 uptake of that observed with AA (Fig. 2). Relatively little O2 uptake, if any, was observed after the addition of ovine COX-1 (oCOX-1) to the reaction containing NAGly. In contrast, COX-1 metabolizes AEA and 2-AG, but still to a much lesser extent than AA. These results show that NAGly is the most selective substrate identified to date. Steady-state kinetic analysis revealed that the Km for
Discussion
The present studies establish a possible role for COX-2 in the metabolism and regulation of the anti-inflammatory lipoamino acid, NAGly. COX-2 oxygenates NAGly with a moderate efficiency, in contrast to the absence of metabolism by COX-1. Ranking the catalytic efficiency of COX-2 substrates from the most to the least efficient (AA>2-AG>NAGly>AEA) suggests that modification of the alcohol group in AEA to the carboxylic acid in NAGly promotes higher affinity and more productive binding for
Acknowledgements
This research was supported in part by National Institutes of Health Research, Center, and Training Grants CA89450, CA09592, and GM15431. The project described was also supported in part by the Molecular Recognition Unit Center grants from the National Cancer Institute (CA68485) and the National Institute of Environmental Health Sciences (P30 ES00267).
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Abbreviations: NAGly, N-arachidonylglycine; AA, arachidonic acid; COX, cyclooxygenase; mCOX-2, murine COX-2; oCOX-1, ovine COX-1; PG, prostaglandin; HETE, hydroxyeicosatetraenoic acid; NSAID, nonsteroidal anti-inflammatory drug; 2-AG, 2-arachidonylglycerol; AEA, anandamide.