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.
Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
T
T
Yamauchi
F
F
Umeda
M
M
Masakado
M
M
Isaji
S
S
Mizushima
H
H
Nawata
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