Purification and Characterization of Recombinant Human Prostacyclin Synthase
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概要
- 論文の詳細を見る
Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H2 to prostacyclin (PGI2), is a member of the cytochrome P-450 (P 450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf 21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf 21 cells after culture in 5 μg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The Km and Vmax values of the purified human PGIS for PGH2 were 30 μM and 15 μmol/min/mg of protein at 24°C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P 450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH2 analogue, U 46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH2 analogue, U 44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys 441 His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P 450, indicating substrate specificity.
- 社団法人 日本生化学会の論文
著者
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Wada Masayuki
Department Of Electrical And Electronic Engineering University Of Miyazaki
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Yokoyama Chieko
Department Of Cellular Physiological Chemistry Tokyo Medical And Dental University
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Ullrich Volker
Department Of Biology University Of Konstanz
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Hatae Toshihisa
Department Of Pharmacology National Cardiovascular Center Research Institute
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Shimonishi Manabu
Department Of Pharmacology National Cardiovascular Center Research Institute
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Imai Yoshio
Department Of Oral Functional Science Graduate School Of Dental Medicine Hokkaido University
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Tanabe Tadashi
Department Of Geriatric Medicine Osaka University Graduate School Of Medicine
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Nakamura Masahiko
Division Of Physiology Institute For Protein Research Osaka University
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