Characterization of a heat-stable fraction of lipovitellin and development of an immunoassay for vitellogenin and yolk protein in winter flounder (Pleuronectes americanus).
An enzyme-linked immunoabsorbent assay was developed for detection and quantification of the yolk protein lipovitellin (Lv) and its plasma precursor, vitellogenin (Vg), in winter flounder (Pleuronectes americanus). Native Lv was found to be a mixture of heat-stable and heat-labile molecules in mature, ovulated eggs. A heat-stable Lv fraction was purified from extracts of unfertilized eggs by brief heat treatment and gel permeation chromatography on Bio-Gel A-1.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat-stable Lv revealed a single polypeptide of 94 kD, while native Lv also possessed several smaller polypeptides, suggesting that heat-labile Lv contains proteolytic cleavages of the 94-kD polypeptide which destabilize its structure. The Stokes radius of the native protein on Bio-Gel A-1.5 was estimated at 4.50 nm, while the Stokes radii of heat-stable and heat-labile Lv were 4.26 nm and 5.17 nm, respectively. Heat-stable Lv was used to produce a rabbit polyclonal antiserum which reacted with a single 175-kD polypeptide in Western blots of vitellogenic female winter flounder serum, but did not react with any component of male serum. Ouchterlony double diffusion using this antiserum demonstrated immunological identity of Lv, heat-stable Lv, and Vg. The anti-Lv anti-serum was used to construct an homologous ELISA with a linear response between 25 and 300 ng/ml. This assay was used to characterize a Bio-Gel A-1.5 column profile of serum from an estradiol-treated male winter flounder, and a single peak, with Stokes radius of 6.70 nm, was identified as Vg. Winter flounder Vg was confirmed to be a dimer, while Lv from mature eggs was found to be a monomer of a lower molecular weight polypeptide.
Hartling
R C
RC
Program in Molecular and Cellular Biology, University of Massachusetts Amherst 01003, USA.
Pereira
J J
JJ
Kunkel
J G
JG
D000818:Animals; D000918:Antibody Specificity; D004527:Egg Proteins / Q000737:chemistry* / Q000276:immunology; D004530:Egg Yolk / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005432:Flounder; D007118:Immunoassay / Q000379:methods*; D005779:Immunodiffusion; D008297:Male; D010063:Ovum / Q000737:chemistry* / Q000276:immunology; D014819:Vitellogenins / Q000032:analysis* / Q000276:immunology
D004527:Egg Proteins; D014819:Vitellogenins