Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.
To purify and characterize the isoform of prostaglandin endoperoxide synthase (rPGSi) induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles, solubilized cell extracts were subjected to anionic exchange chromatography, column fractions were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and rPGSi was visualized by immunoblotting and silver staining techniques. Immunoreactive rPGSi and peroxidase activity co-eluted at pH 6.5 and 6.0. Amino-terminal amino acid sequence of three immunoreactive rPGSi bands (Mr = 72,000, 70,000, and 59,000) were identical, Mr = 59,000 being a proteolytic fragment. Alignment of the amino-terminal sequences of rPGSi with those of ovine PGS (oPGS) indicated that only 15 of 26 residues were identical (58% identity). In contrast, rPGSi was closely related to the deduced amino acid sequence of PGS-related cDNA clones isolated from chicken and mouse cell lines, with 77% (20/26 residues) and 96% (25/26 residues) identity, respectively. Whereas tryptic digests of oPGS generated fragments of Mr = 38,000 and Mr = 33,000, only a small peptide appeared cleaved from the carboxyl terminus of rPGSi. Peroxidase activity of partially purified rPGSi exhibited lower apparent Km and maximal velocity than that of oPGS. Collectively, these results document the existence of a novel rat PGS isoform (based on purification, enzymatic activity, and amino-terminal amino acid sequence) which is hormonally induced and obligatory for a known biological process, ovulation.
Sirois
J
J
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Richards
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D007016:Hypophysectomy; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification*; D007668:Kidney / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010060:Ovulation / Q000187:drug effects; D010449:Peptide Mapping; D011450:Prostaglandin Endoperoxides, Synthetic / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014599:Uterus / Q000187:drug effects / Q000201:enzymology
D006063:Chorionic Gonadotropin; D007527:Isoenzymes; D011450:Prostaglandin Endoperoxides, Synthetic; D004958:Estradiol; D005640:Follicle Stimulating Hormone