Purification and molecular cloning of prostacyclin-stimulating factor from serum-free conditioned medium of human diploid fibroblast cells.
We attempted to identify the factor that stimulated prostacyclin (PGI2) production using conditioned medium from cultured human diploid fibroblast cells subjected to a series of purification steps using h.p.l.c. on DEAE-5PW, Heparin-5PW, Protein-Pak 300, and an insulin-like growth factor-1 ligand affinity column. The purified prostacyclin-stimulating factor (PSF) ran as a single band with a molecular mass of 31 kDa by SDS/PAGE. Analysis of the purified PSF by C4 reversed-phase h.p.l.c. showed a single sharp peak in 31% (v/v) acetonitrile. The material was purified 8000-fold with an overall yield of about 18%. The purified PSF stimulated PGI2 production by cultured bovine aortic endothelial cells at a concentration of about 10 ng/ml; maximal stimulation was achieved at a concentration of 25 ng/ml. A cDNA coding for PSF was cloned and sequenced, revealing an apparently novel protein with no obvious sequence similarity to known proteins.
Yamauchi
T
T
Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Umeda
F
F
Masakado
M
M
Isaji
M
M
Mizushima
S
S
Nawata
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001685:Biological Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004171:Diploidy; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011464:Epoprostenol / Q000096:biosynthesis*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data
D001685:Biological Factors; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; C057670:prostacyclin synthesis stimulating plasma factor; D011464:Epoprostenol