Characterization and quantification of yolk proteins in the lobster, Homarus americanus.
Yolk protein (vitellin, Vn) and its precursor (vitellogenin, Vg) were isolated and characterized in the ovary and hemolymph, respectively, of the adult female lobster, Homarus americanus. Vn had a molecular mass of 360 kDa when analyzed by gel filtration. When analyzed by SDS-PAGE, Vn had six bands (110, 105, 94, 90, 81, and 78 kDa). An anti-Vn antiserum was developed using purified Vn, and the antiserum was used to detect Vn and Vg by ELISA and western blot techniques. ELISA analysis of hemolymph proteins separated by gel filtration indicated that Vg was similar in mass to Vn (360 kDa). However, western blots of hemolymph proteins separated by SDS-PAGE indicated that Vg contained a pair of protein subunits, 194 kDa and 179 kDa. Furthermore, the elution profiles of Vn and Vg from anion exchange chromatography indicated that Vg had a more negative charge. Thus, Vg appears to be processed after its uptake by the ovary to form Vn. Vg was undetectable in hemolymph from adult males by either ELISA or by western blot analysis. However, hemolymph levels of Vg in adult females increased 40-fold during the reproductive cycle, rising from 18 microg/mL in ovarian stage II to 789 microg/mL at stage V. This increase correlates well with oocyte growth during the cycle. Hence, this method may be useful for studying the regulation of lobster vitellogenesis.
Tsukimura
Brian
B
Department of Biology, California State University, Fresno, California 93740, USA. BrianT@CSUF resno.edu
Waddy
Susan L
SL
Vogel
Jacalyn M
JM
Linder
Colby J
CJ
Bauer
Daniel K
DK
Borst
David W
DW
D000818:Animals; D015153:Blotting, Western; D004527:Egg Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006458:Hemolymph / Q000737:chemistry; D008121:Nephropidae / Q000502:physiology*; D009865:Oocytes / Q000254:growth & development; D010053:Ovary / Q000737:chemistry; D014818:Vitellogenesis / Q000502:physiology*
D004527:Egg Proteins