出版社：American Society for Biochemistry and Molecular Biology
摘要：The mechanism by which the inflammatory enzyme prostaglandinH2 synthase-1 (PGHS-1) deactivates remains undefined. This studyaimed to determine the stabilizing parameters of PGHS-1 andidentify factors leading to deactivation by nitric oxide species(NOx). Purified PGHS-1 was stabilized when solubilized in ß-octylglucoside(rather than Tween-20 or CHAPS) and when reconstituted withhemin chloride (rather than hematin). Peroxynitrite (ONOO–)activated the peroxidase site of PGHS-1 independently of thecyclooxygenase site. After ONOO– exposure, holoPGHS-1could not metabolize arachidonic acid and was structurally compromised,whereas apoPGHS-1 retained full activity once reconstitutedwith heme. After incubation of holoPGHS-1 with ONOO–,heme absorbance was diminished but to a lesser extent than theloss in enzymatic function, suggesting the contribution of morethan one process to enzyme inactivation. Hydroperoxide scavengersimproved enzyme activity, whereas hydroxyl radical scavengersprovided no protection from the effects of ONOO–. Massspectral analyses revealed that tyrosine 385 (Tyr 385) is atarget for nitration by ONOO– only when heme is present.Multimer formation was also observed and required heme but couldbe attenuated by arachidonic acid substrate. We conclude thatthe heme plays a role in catalyzing Tyr 385 nitration by ONOO–and the demise of PGHS-1.Supplementary key words cyclooxygenase • eicosanoids • heme reconstitution • peroxidase activity • proteolysis • mass spectrometry
Abbreviations: C10M, N-decyl-ß-D-maltopyranoside; DEDTC, diethyldithiocarbamate; , extinction coefficient; EtOH, ethanol; KI, potassium iodide; L-NAME, NG-nitro-L-arginine methyl ester; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; nLC-MS/MS, nanoflow liquid chromatography-tandem mass spectrometry; NOx, nitric oxide species; NOC-7, 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene; ßOG, N-octyl-ß-D-glucopyranoside; •OH, hydroxyl radical; ONOO–, peroxynitrite; PGHS-1, prostaglandin H2 synthase-1; SIN-1, 3-morpholinosydnonimine hydrochloride; TMPD, N,N,N',N'-tetramethylphenylenediamine; TNM, tetranitromethane; Tyr 385, tyrosine 385; UV-Vis, ultraviolet-visible
, extinction coefficient; EtOH, ethanol; KI, potassium iodide; L-NAME, NG-nitro-L-arginine methyl ester; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; nLC-MS/MS, nanoflow liquid chromatography-tandem mass spectrometry; NOx, nitric oxide species; NOC-7, 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene; ßOG, N-octyl-ß-D-glucopyranoside; •OH, hydroxyl radical; ONOO–, peroxynitrite; PGHS-1, prostaglandin H2 synthase-1; SIN-1, 3-morpholinosydnonimine hydrochloride; TMPD, N,N,N',N'-tetramethylphenylenediamine; TNM, tetranitromethane; Tyr 385, tyrosine 385; UV-Vis, ultraviolet-visible
