ABSTRACT
Estrogen treatment of medaka leads to accumulation of ascites, in which vitellogenin (Vg) and choriogenins (precursors to vitelline envelope) are abundant. Besides those female-specific proteins, we detected a new component in ascites that cross-reacts with antiserum against egg yolk proteins. We tentatively named it egg yolk-related protein (YRP). YRP was purified from ascites by hydroxylapatite chromatography followed by gel filtration. Purified YRP had a molecular mass of 460 kDa in intact state while 570 kDa for Vg. The molecular weight of purified YRP on SDS-PAGE under both reducing and nonreducing conditions was 130 kDa. YRP was confirmed to be a lipoglycophosphoprotein by staining with Sudan black, periodic acid-Schiff (PAS) and methyl green. Amino acid composition of YRP resembled that of Vg except for a relatively low content of serine. A specific antiserum against YRP was raised in a rabbit. Antiserum against YRP specifically immunostained its antigen but not Vg or choriogenins. YRP was detected as a female-specific protein in serum of breeding medaka. The antiserum also cross-reacted with a band at 29 kDa in egg extracts, which is not immunoreactive to antiserum against Vg. These data show that YRP is a precursor to some egg yolk proteins with differing antigenicity from Vg (Hamazaki et al. '87). We thus conclude that YRP is a second form of medaka Vg and rename YRP as Vg 2 while formerly reported Vg as Vg 1.
Subject(s)
Ascitic Fluid/chemistry , Estrogens/pharmacology , Oryzias/physiology , Vitellogenins/analogs & derivatives , Vitellogenins/isolation & purification , Amino Acids/analysis , Animals , Antibody Specificity , Blotting, Western , Egg Proteins/chemistry , Egg Proteins/immunology , Electrophoresis, Polyacrylamide Gel , Estrogens/administration & dosage , Female , Immunodiffusion , Male , Molecular Weight , Vitellogenins/chemistry , Vitellogenins/immunologyABSTRACT
A simple procedure for radiolabeling of locust vitellogenin is described. This procedure involves coupling of [3H]propionyl succinimidate to purified vitellogenin with high yield and specific activity. Using this radiolabeled analog, a specific and sensitive radioimmunoassay was developed for determining locust vitellogenin content, with a lower detection limit of 1 ng. [3H]Propionyl-vitellogenin binds completely to rabbit anti-vitellogenin (locust) and can be completely competed out by locust vitellogenin. The structural similarity of locust vitellogenin with that of locust egg vitellin, male locust lipophorin (a diglyceride-carrying lipoprotein), Xenopus laevis vitellogenin, and chicken egg yolk lipovitellin was examined with this RIA procedure. Comparable binding competition was obtained with locust vitellin only. Male locust lipophorin, Xenopus vitellogenin, and chicken lipovitellin did not inhibit vitellogenin binding at concentrations 1000-fold greater than that of locust vitellogenin. The use of this RIA in determination of vitellogenin synthesis in vivo and in vitro, using isolated fat body preparations, is described.
