Purification, Characterization, Immunoassay And Function Study Of Vitellogenin And Lipovitellin From Rosy Barb (Puntius Conchonius)

Vitellogenin (Vg) is the egg yolk protein precursor in all egg-laying animals. It is a high molecular mass phospholipoglycoprotein produced in response to estrogen stimulation. Lipovitellin (Lv) is one of the major proteolytic products of vitellogenin after it is taken up by growing ovary in the manner of receptor-mediated endocytosis, which provides nutrient storage for the developing embryos and larvae. Recent study also shows that Vg and Lv have a potential use. The rosy barb (Puntius conchonius), as a experimental fish widely used in ecotoxicological study, has not been reported for the structural and functional study of vitellogenin and lipovitellin. In this paper, Vg and Lv were purified by double-step chromatography from mature female rosy barb plasma and from their ovaries, respectively. The Vg appeared to exist as a homotrimer of approximately 450 kDa in native polyacrylamide gel electrophoresis (PAGE), and was reduced to a single monomer of approximately 167 kDa in SDS-PAGE. The Lv was a 370 kDa heterogenous trimer and was reduced to three subunits with molecular mass of 125, 110, 76 kDa in SDS-PAGE, which was firstly reported in the teleost. The Vg was characterized as a phospholipoglycoprotein by native PAGE and staining of gels for phosphorus with methyl green, for lipds with sudan black B, and for carbohydrates with periodic acid-Schiff reagent. The Lv was similarly characterized as a lipoglycoprotein. Analysis of the amino-acid composition of rosy barb Vg revealed the presence of relatively high levels of Ala (10.58%), Asp (8.46%), Glu (10.30%), Leu (11.23%), Lys (7.22%) and Val (7.49%), accounting for 55.28% by weight of the total amino acids present. However, levels of His (2.76%), Met (2.05%) and Tyr (3.43%) for rosy barb Vg were relatively lower. These were closely similar to those reported for other teleosts. Lv was rich in Ala (12.19%),Asp (7.56%U}lu(11.63%) .Leu (11.05%) and Val (7.90%) . accounting for 50.33% bvweight of the total amino acids, and contained low levels of His (2.54%) , Met (2.44%) and Tyr (2.39%), accounting for 7.37% by weight of the total amino acids. All these indicate that the purified proteins resemble Vg and Lv in other teleostean species and therefore are reasonably identified as rosy barb Vg and Lv.Polyclonal antisera against the purified Vg and Lv from rosy barb were raised in mice. Double immunodiffusion showed determination of the litre for Vg and Lv antisera were 1:32 and above 1:64 respectively. Double immunodiffusion assay showed that the plasma and ovary extract from the female rosy barb reacted with the mouse antisera against rosy barb Vg, forming a single immunoprecipitin line, while the plasma from the male rosy barb or from the female zebrafish did not, confirmingthe normal existence of the sex- and species-specific reactivity for rosy barb Vg.Native PAGE and Western blotting analyses demonstrated that both plasma and the ovary extract from the sexually-mature female rosy barb cross-reacted with the mouse Lv antisera. In double immunodiffusion, the female rosy barb muscle extract, male rosy barb testis and muscle extracts, female zebrafish ovary extract, or plasma from both male rosy barb and female zebrafish did not react with Lv antisera. These indicated that rosy barb Lv is species-specific, female-specific and tissue-specific protein, and Lv has common antigenicity with its precursor Vg.Vg and Lv could only agglutinate the chick erythrocytes intensively and selectively, the hemagglutination (HA) liters of Vg and Lv were 2"9 and 2"8. It was showed for the first time that teleostean Vg and Lv had hemagglutinating activities. The hemagglutinating activities of Vg and Lv were sensitive to high temperature and stable in neutral state. The hemagglutinating activity of Vg and Lv were not affected by treatment with SDS and P -ME. The hemagglutinating activity of Vg was affected by treatment with CaCl2 and EDTA, which suggested that the hemagglutinating activity of Vg is Ca2+-dependant, but Lv is not obvious. The different destructive treatments of Vg a...