Purification and characterization of fatty acid cyclooxygenase from human platelets.
The fatty acid cyclooxygenase (EC 1.14.99.1) that produces the prostaglandin, thromboxane, and prostacyclin precursor (PGH2), was solubilized from human platelet microsomes in 20 sucrose and 1.0% Triton X-100. The enzyme was purified 300-fold by electrofocusing, Sephadex G-200 gel filtration, and hydrophobic chromatography on ethyl agarose. The cyclooxygenase catalyzed the conversion of arachidonic acid to prostaglandin endoperioxide, PGH2, that was trapped at -25 degrees C and separated on TLC at -20 degrees C. PGH2 was hydrolyzed to HHT in acidic pH, or was chemically converted to PGE2 in slightly alkaline pH in the absence of cofactors. The enzyme showed a broad pH optimum in the range of 7-9. Hemin containing substances such as methemoglobin were absolutely required as cofactors, while tryptophan, epinephrine, phenol, and hydroquinone stimulated the PGH2 formation. Metal ions, such as ZN2+ and Cd2+ inhibited the enzyme reaction at 0.1 to 1 mM. The molecular weight of the purified enzyme was estimated at 79,432 by sodium dodecyl sulfate disc gel electrophoresis at pH 8.0. The properties of the human platelet enzyme was generally similar to the sheep vesicular enzyme in the method of solubilization, pH optimum, and molecular weight.
D001792:Blood Platelets / Q000201:enzymology*; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D006801:Humans; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011451:Prostaglandin-Endoperoxide Synthases / Q000097:blood / Q000302:isolation & purification*; D011463:Prostaglandins H / Q000378:metabolism; D014462:Ultrafiltration
D011463:Prostaglandins H; D011451:Prostaglandin-Endoperoxide Synthases