Purification and characterization of recombinant human prostacyclin synthase.
Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/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 K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 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 PGH(2) analogue, U46619, 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 PGH(2) analogue, U44069, 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 Cys441His 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 P450, indicating substrate specificity.
Wada
Masayuki
M
Department of Pharmacology, National Cardiovascular Center Research Institute and Division of Microcirculatory Kinetics, Osaka University Graduate School of Medicine, National Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565.
Yokoyama
Chieko
C
Hatae
Toshihisa
T
Shimonishi
Manabu
M
Nakamura
Masahiko
M
Imai
Yoshio
Y
Ullrich
Volker
V
Tanabe
Tadashi
T
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000737:chemistry; D015121:6-Ketoprostaglandin F1 alpha / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017886:Durapatite / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D010084:Oxidation-Reduction; D017354:Point Mutation; D011450:Prostaglandin Endoperoxides, Synthetic / Q000737:chemistry; D044262:Prostaglandin H2 / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry; D018411:Spodoptera; D013928:Thromboxane A2 / Q000031:analogs & derivatives; D014162:Transfection; D014191:Tranylcypromine / Q000737:chemistry
D011450:Prostaglandin Endoperoxides, Synthetic; D011994:Recombinant Proteins; D014191:Tranylcypromine; D044262:Prostaglandin H2; C028429:U 44069; D013928:Thromboxane A2; D015121:6-Ketoprostaglandin F1 alpha; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase