Purification of mummichog (Fundulus heteroclitus) gonadotropins and their subunits, using an immunochemical assay with antisera raised against synthetic peptides.
To detect mummichog gonadotropins (GtHs) and their subunits immunochemically, fragment peptides with amino acid sequences corresponding to cDNA data were synthesized, and antisera were raised against them. In the case of GtH-IIbeta, large loops such as the second loop and the "seat belt" structure (deduced from the hCG 3D structural data) were considered to be favorable regions for antigen, although further examination is needed to determine if this is the case of GtH-Ibeta and GtH-alpha. In the purification process, glycoprotein was extracted from acetone-dried mummichog pituitary and separated by various liquid chromatography procedures. Each fraction was assayed by immunoblotting with the appropriate antisera against synthetic peptides. Subunits (GtH-alpha, GtH-Ibeta, and GtH-IIbeta) were obtained through gel filtration, anion-exchange chromatography, and reverse-phase HPLC. Intact bioactive GtH-I and GtH-II were obtained through gel filtration, anion-exchange chromatography, and hydrophobic chromatography. Both GtH-I and GtH-II dissociated into subunits under acidic conditions. Nominal MW of each subunit was estimated from SDS-PAGE as 23,000 for GtH-alpha from GtH-I, 22,000 for GtH-alpha from GtH-II, 18,000 for GtH-Ibeta, and 21,000 for GtH-IIbeta.
Shimizu
Akio
A
National Research Institute of Fisheries Science, Fukuura 2-12-4, Kanazawa, Yokohama 236-8648, Japan.
Yamashita
Michiaki
M
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D023781:Fundulidae / Q000378:metabolism*; D006031:Glycosylation; D006062:Gonadotropins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D007120:Immunochemistry; D008297:Male; D008969:Molecular Sequence Data; D010053:Ovary / Q000187:drug effects; D010455:Peptides / Q000276:immunology*; D010902:Pituitary Gland / Q000737:chemistry
D006062:Gonadotropins; D010455:Peptides