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Purification of multiple vitellogenins in grey mullet (Mugil cephalus)

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Abstract

Three female specific serum proteins were detected immunologically in the sera of grey mullet (Mugil cephalus) which were named vitellogenin A (VgA), VgB, and VgC, based upon their distinct antigenicity against specific antisera raised against three types of mullet lipovitellins (Lvs). These Vgs were subsequently purified from the serum of estradiol-treated mullet by combining several types of chromatography columns (anion exchanger, hydroxylapatite, immunoadsorbent column, and gel filtration). Purified native VgA, VgB, and VgC exhibited molecular masses of 570, 580, and 335 kDa, respectively. Following, SDS-PAGE, the estimated mass of polypeptide bands evident for VgA and VgB were ∼179 and ∼175 kDa, respectively; VgC appeared to be ∼132 kDa. The two larger Vgs (VgA and VgB) appeared to be phosphorylated, suggesting that these Vgs contain a highly phosphorylated, serine-rich phosvitin (Pv) domain. Furthermore, two discrete Vg-type specific antisera, anti-VgA and anti-VgB, were developed and each generated two precipitin lines against ovary extracts in immunoelectrophoresis, indicating that these Vgs contain additional antigenic yolk protein domains: Lv and β′-component. The small Vg (VgC) appeared to lack a Pv domain because of its low serine content (5.35%) and failure to show positive results in phospho-staining experiments. In conjunction with N-terminal amino acid sequencing analyses of the purified Vgs, our present results have conclusively identified the purified Vg products in grey mullet as typical A-type (VgA), B-type (VgB), and C-type (VgC) Vgs.

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Acknowledgments

This study was supported in part by the Grants-in Aid for twenty-first century COE Program (#16COE02217) and Scientific Research (#1778014806, #19380106, and #19780143) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. All experiments complied with the current laws of Japan. We thank Associate Professor Kiyoshi Soyano, Institute for East China Sea Research, Nagasaki University and Professor Hirohiko Kagawa, Faculty of Agriculture, University of Miyazaki for kindly providing mullet sera.

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Authors and Affiliations

  1. Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, 041-8611, Japan

    Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu & Akihiko Hara

  2. Department of Zoology, North Carolina State University, Raleigh, NC, 27695-7617, USA

    Naoshi Hiramatsu & Craig V. Sullivan

  3. Hokkaido National Fisheries Research Institute, Fisheries Research Agency, Kushiro, Hokkaido, 085-0802, Japan

    Sayumi Sawaguchi & Takahiro Matsubara

Authors
  1. Haruna Amano
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  2. Toshiaki Fujita
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  3. Naoshi Hiramatsu
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  4. Sayumi Sawaguchi
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  5. Takahiro Matsubara
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  6. Craig V. Sullivan
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  7. Akihiko Hara
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Correspondence to Akihiko Hara.

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Communicated by S. Nishida.

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Amano, H., Fujita, T., Hiramatsu, N. et al. Purification of multiple vitellogenins in grey mullet (Mugil cephalus). Mar Biol 152, 1215–1225 (2007). https://doi.org/10.1007/s00227-007-0768-z

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  • DOI: https://doi.org/10.1007/s00227-007-0768-z

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