類卵黏質醣蛋白是大頭鰱未受精卵外膜的主要成分

Abstract

大頭鰱（Hypophthalmichthys nobilis, Bighead Carp）未受精卵在含有protease inhibitors的100mM EDTA, 500mM NaCl, Tris•HCl（PH=8.5）溶液中磨碎。磨碎後得到的卵鞘（egg shells）通過兩個重疊的篩子(上層篩孔直徑180um，下層篩孔直徑63um）。以含有8M urea, l% SDS, 30mM DTT, 10mM EDTA的Tris•HCl ( PH=8.5）溶液在70℃水浴溶解下層篩子得到的卵鞘。經SDS- PAGE (6% acrylaoide)及PAS staining發現溶解出來的蛋白質主要是分子量46Kd及 92Kd的兩種醣蛋白。 以electroelution純化46Kd和92Kd醣蛋白，將二者分別跑電泳，發現46Kd糖蛋白在電泳片46Kd的位置出現major band, 在92Kd的位置出現minor band。92Kd醣蛋白的情形則相反。Western blot顯示二者皆與92Kd的抗體結合。二者N端10個胺基酸幾乎一樣，而且胺基酸組成也非常接近。由以上實驗結果証明92Kd醣蛋白是46Kd醣蛋白的雙倍體(dimer）。從protein data bank發現此種醣蛋白和雞蛋白的卵黏質醣蛋白(ovomucoid glycoprotein) N端15個胺基酸非常相似，但是二者胺基酸組成有很大的差異。因此46Kd和 92Kd醣蛋白是一種類卵黏質醣蛋白。 Western blot analysis証明在母大頭鰱其他組織沒有類卵黏質醣蛋白。在大頭鰱卵外膜和卵內含物的Western blot analysis發現類卵黏質醣蛋白得自卵外膜而且是卵外膜的主要成分。用Western blot分析取魚卵外膜和卵內含物，發現鯉魚卵外膜主要醣蛋白和大頭鰱卵外膜主要醣蛋白不同。雞蛋白的卵黏質（ovomucoid）是一種trypsin inhibitor 。至目前為止，大頭鰱卵外膜類卵黏質醣蛋白沒有trypsin-inhibition的活性。

Patterns of SDS-PAGE (6% acrylamide) show that bighead carp unfertilized eggshell has two major bands corresponding to 92Kd and 46Kd. Both bands showed PAS stain positive. Purify 46Kd and 92Kd glycoproteins by electroelution. After SDS-PAGE 46Kd glycoprotein appeared as the major band at 46Kd and a minor band at 92Kd position. As to 92Kd glycoprotein, it behaved in a similar way. Western blot shows that both proteins could be bound by antibody of 92Kd. N-terminal 10 amino acids of 92Kd and 46Kd glycoproteins are the same, and amino acid compositions of both proteins are almost the same. Above experimental results prove that 92Kd glycoprotein is the dimer of 46Kd glycoprotein. N-terminal 15 amino acids of 46Kd glycoprotein and chicken egg white ovomucoid glycoprotein are almost the same, but amino acid compositions of both proteins are quite different. 46Kd glycoprotein is regarded as ovomucoid-like glycoprotein. In western blot we found that there is no binding by antibody of 92Kd in other tissues of female bighead carp. Western blot of bighead carp egg envelope and contents within egg shows that 92Kd and 46Kd glycoproteins derived exclusively from the egg envelope and are the major components of egg envelope. We also found that carp egg envelope major glycoprotein is different from bighead carp by Western blot analysis. Chicken ovornucoid is a kind of trypsin inhibitor. So far the ovomucoid-like glycoprotein from bighead carp egg envelope has apparently no trypsin- inhibition activity.