The purification and partial characterization of carp, Cyprinus carpio, vitellogenin

Abstract

A procedure is described for the isolation of intact vitellogenin (c-VTG) from the carp, Cyprinus carpio. VTG was induced in juvenile females using oestradiol-17β and purified from the plasma using a combination of gel-filtration chromatography on Sepharose 6B and ion exchange chromatography on DEAE-cellulose. Purification procedures were conducted at low temperatures (below 9°C) in the presence of the proteolytic enzyme inhibitor aprotinin to prevent degradation. Intact c-VTG had an apparent molecular mass of 390,000 Daltons, but when extracted from plasma in the absence of aprotinin it underwent proteolysis into at least 2 protein fragments (apparent molecular masses of 230,000 and 96,000 Daltons), showing an instability of the native dimer. An amino acid analysis of c-VTG showed that its composition was almost identical to goldfish VTG, a species closely allied to the true carps and also similar to other oviparous vertebrate VTGs. Collectively, these data indicate that using these purification procedures VTG from carp, and probably other teleost species, can be isolated in an intact, highly purified form.

Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction, isolation and partial characterization

“…E 2 induces the synthesis of VTG in a number of teleost species, including goldfish, rainbow trout, sea trout, Atlantic salmon, turbot, carp, striped bass, Atlantic cod, striped wolffish and sea bass (de Vlaming et al 1980;Norberg and Haux 1985;Olin and von der Decken 1987;Silversand and Haux 1989;Tyler and Sumpter 1990;Kishida et al 1992;Silversand et al 1993;Mafian6s et al 1994). Recently, E 2 has been found to induce hepatic synthesis of other components of eggs from teleosts, including Atlantic halibut, such as vitelline envelope proteins (Hyllner et al 1991(Hyllner et al , 1994.…”

“…Fish reproducing in fresh water, such as goldfish (Carassius auratus;de Vlaming et al 1980), rainbow trout (Oncorhynchus mykiss; Campbell and Idler 1980;Norberg and Haux 1985), catfish (Heteropneustes fossilis; Nath and Sundararaj 1981), sea trout (Salmo trutta; Norberg and Haux 1985), carp (Carassius carassius; Tyler and Sumpter 1990) and striped bass (Morone saxatilis; Kishida et al 1992) generally have dimeric VTGs with molecular masses around 400 kDa, a lipid content around 20% and a phosphorus content around 0.6%. In coldwater marine fish, VTG is less studied.…”

Measurement of vitellogenin, a biomarker for exposure to oestrogenic chemicals, in a wide variety of cyprinid fish

“…Blood was collected 7 days after the second injection of oestradiol-17fi and the resulting plasma was subjected to size-exclusion filtration (using 100kDa molecular weight cut-off membranes; Amicon) followed by ion-exchange chromatography on DEAE-cellulose and, finally, a second 100 kDa size-exclusion filtration. The c-VTG obtained was considered to be highly purified and eluted as a single, well-defined peak when run on a 1-m column of Sepharose-6B (Tyler and Sumpter 1990a),…”

“…Carp vitellogenin was purified from plasma using a procedure similar to that described in Tyler and Sumpter (1990a). Briefly, VTG synthesis was induced in an adult female mirror carp using two intraperitoneal injections of oestradiol-17fl (1 mg'kg -1 body weight), spaced 7 days apart.…”

Sexual maturity in sea trout, Salmo trutta L., running up the River Calonne (Normandy, France) at the ‘finnock’ stage

“…The blood was collected, centrifuged and the plasma stored at -20°C until use. VTG was purified according to Tyler & Sumpter (1990). Purified VTG was injected over a period of 4 months (eight injections of lOOpg) into rabbits.…”