Purification of prostacyclin synthase from bovine aorta by immunoaffinity chromatography. Evidence that the enzyme is a hemoprotein.

Two mouse spleen cell-myeloma cell hybridomas (isn-1 and isn-3) which secrete IgGl molecules capable of precipitating prostaglandin I2 (PGIZ) synthase activity were selected and cloned. Immunoglobulins secreted by isn-1 and isn-3, but not by control hybridoma lines, caused the immunoprecipitation of the same, single ‘2sI-labeled protein (Mr = 52,000) from a mixture of 12SI-labeled proteins from bovine aortic microsomes. IgGl (isn-3) was coupled to Affi-Gel 10 and used to purify PG12 synthase activity in 20% yield from bovine aortic microsomes solubilized with Triton X-100. The purified synthase exhibited only one protein band (Mr = 52,000) when examined by sodium dodecyl sulfategel electrophoresis. At a PGH2 concentration of 50 p ~ , the homogeneous enzyme preparation catalyzed the formation of approximately 1,000 nmol of PGIz/min/ mg of protein at 24 “C. The visible absorption spectrum of purified PGIz synthase exhibited a major heme peak at 418-420 nm; the difference spectrum of enzyme treated with sodium dithionite and CO exhibited a broad absorption maximum centered around 440 nm. Purified PGIz synthase was inactivated rapidly during incubation with substrate PGH2 or with 13-hydroperoxylinoleic acid; loss of enzyme activity was associated with bleaching of the major heme absorption peak. The losses of enzyme activity and heme absorbance induced by either PGHz or 13-hydroperoxylinoleate were prevented when the PGHz analog 9,11-azoprosta-5,13-dienoic acid was included at inhibitory concentrations in the incubation mixture. Our results indicate that PGI2 synthase is a ferrihemoprotein with a subunit molecular mass of 52,000 daltons, and that the enzyme is inactivated directly by treatment with alkyl hydroperoxides and during the conversion of PGH2 to PG12. The correlation between the bleaching of the heme spectrum and the loss of enzyme activity and the prevention of both these phenomena by a nonreactive endoperoxide analog suggests that a ferriheme at the active site of the enzyme participates in cleavage of the endoperoxide group of PGHz leading to prostacyclin formation.