title: Purification of multiple precursors for egg chorion proteins in Atlantic cod (Gadus morhua).
abstract: Egg chorion precursors (zona radiata proteins; Zrps) were purified from the blood plasma of female Atlantic cod (Gadus morhua) by salting-out and column chromatography. The salting-out procedure employed a relatively low (30%) concentration of saturated ammonium sulfate. This was a critical step that separated Zrps from approximately 89% of other plasma proteins. Subsequently, three subtypes of Zrp (Zrp-alpha, -beta and -gamma) were purified by four (Zrps-alpha, -gamma) or five (Zrp-beta) serial column chromatography steps. The Intact masses of purified Zrp-alpha, -beta and -gamma were 290 kDa, 134 kDa, and 73 kDa, while masses estimated by SDS-PAGE were 78 kDa, 54 kDa, and 47 kDa, respectively. Antibodies were prepared against Zrp-beta and -gamma and utilized to develop specific immunoassays. The plasma levels of Zrp-beta and -gamma In reproductive female cod were estimated to be 591.42+/-77.59 microg/ml and 768.71+/-120.39 microg/ml, respectively. Thus, practical procedures for the separation of Zrp subtypes were developed in cod, which resulted in the development of subtype-specific Zrp immunoassays in this species; a similar method could be adopted for the separation, detection, and quantification of Zrp subtypes in other teleosts.
authors.affiliation: Division of Marine Biosciences, Faculty of Fisheries, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido, 041-8611, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Fujita
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Scott
authors.forename: Loanna
authors.initials: L
authors.lastname: Katsiadaki
authors.forename: Haruna
authors.initials: H
authors.lastname: Amano
authors.forename: Lei
authors.initials: L
authors.lastname: Hong
authors.forename: Naoshi
authors.initials: N
authors.lastname: Hiramatsu
authors.forename: Takashi
authors.initials: T
authors.lastname: Todo
authors.forename: Akihiko
authors.initials: A
authors.lastname: Hara
mesh_terms: D000818:Animals; D004527:Egg Proteins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D005260:Female; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D048389:Gadus morhua / Q000097:blood / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D007120:Immunochemistry; D010063:Ovum / Q000378:metabolism*; D011498:Protein Precursors / Q000097:blood / Q000378:metabolism*
chemical_list: D004527:Egg Proteins; D029941:Fish Proteins; D011498:Protein Precursors; C423083:zona radiata protein, fish