卤虫（Artemia Shrimp）孵化酶的生物化学特性及孵化腺细胞的免疫细胞化学研究
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摘要
卤虫孵化酶(HE)是由卤虫早期胚胎特异性分泌的、在孵化过程中起关键作用的一种蛋白酶。我们利用67％硫酸铵沉淀、DEAE-sepharose Fast Flow阴离子交换柱层析和Sephacryl凝胶过滤柱层析，并以酪蛋白为其蛋白酶水解活性的检测底物、以卵膜为其卵壳裂解活性的特异性底物，从卤虫胚胎孵化液中分离纯化出了卤虫的孵化酶分子，其在SDS-PAGE电泳中的分子量约为98.5kDa。对孵化酶纯化样品进行生化性质和酶性质分析发现，卤虫孵化酶的最适反应温度约为40℃，最适pH为8.5左右；该孵化酶对p-APMSF、SBTI极为敏感，对PMSF、LBTI和TLCK也非常敏感，但对chymostatin、Leupeptin、Pepstatin、Bestatin、TPCK、NEM和IAM不敏感，表明该酶极可能是一种属于胰蛋白酶类型的丝氨酸蛋白酶。利用EDTA和几种金属离子对该酶活性进行研究发现，该孵化酶可浓度依赖性地为EDTA所抑制，50mmol/L的EDTA对该酶活性的抑制作用可达约56.5％；此外，该酶对Zn~(2+)非常敏感，2.5mmol/L Zn~(2+)可使HE的活性提高为原来的215％，而其它金属离子如Ca~(2+)、Mg~(2+)和Cu~(2+)对该酶活性没有影响或影响不显著，表明卤虫孵化酶很可能又是一种Zn-金属蛋白酶。
免疫细胞化学染色结果显示，卤虫HGC最早出现于孵化前2h，于孵化后5h消失。卤虫HGC于孵化前2h刚刚出现时，胚胎中的HE颗粒界限不明显且数量较少，颜色也很淡，遍布于整个胚体；随胚胎的进一步发育，HGC的数量逐渐增多、颜色也逐渐变深，在孵化时达到最大，之后颗粒量便开始逐渐减少、颜色也逐渐变浅；孵化后2h的幼体中，HE阳性反应减弱，于胚胎局部已开始消失，且主要集中在胚胎中央及胚胎体表，最终于孵化后5h小时消失。卤虫HGC最初出现至孵化前1h时均为全身性分布，从孵出到孵出后2h，头
卤虫孵化酶的生物化学性质及孵化腺细胞的免疫组织化学研究
部的孵化酶颗粒已经减少，而变为非全身性分布，到孵出后sh，孵化酶颗粒
已基本消失殆尽。卤虫胚胎的头部很可能是孵化酶的最早开始分泌的部位，可
能也是主要的分泌部位，随着胚胎的继续孵出，身体背部继而腹部等部位也开
始分泌孵化酶，以协助胚胎完全孵出。孵出后仍残留的孵化酶有可能参与了卤
虫幼体内残余卵黄颗粒的消化降解，此结论还需进一步验证。
The hatching enzyme from brine shrimp, Artemia shrimp, is a pivotal protease which help the encysted embryo escape from its hatching membrane when hatching. The brine shrimp HE was prepared and purified by 67% ammonium sulfate precipitation, DEAE-sepharose Fast Flow ion-exchange chromatography, and Sephacryl column gel-filtration, and its biochemical and enzymological properties were identified in this study. It was found that the deduced molecular weight of HE in SDS-PAGE is about 98.5 kDa, and its proteolytic activity was optimized at pH of 7.5-8.5 and at temperature of 40 , respectively. The HE was strongly inhibited by SBTI and APMSF, and very sensitive to PMSF, LBTI, and TLCK, while not sensitive to chymostatin, bestatin, leupeptin,TPCK, pepstatin, NEM and IAM. All these results imply that brine shrimp HE was most probably a trypsin-type serine protease. The HE could be strongly inhibited by EDTA in a dose-dependent manner, and 50 mmol/L EDTA exhibited more than 56.5% inhibition. The brine shrimp HE w
as extremely sensitive to Zn2+, and 2.5 mmol/L Zn2+ could elevate HE activity for nearly 215 %, the HE was almost unaffected by Ca2+, Mg2+ and Cu2+. It can be concluded that the brine shrimp HE was propably a kind of Zn-metalloprotease.
The immunocytochemical staining methods employing anti-GST-UVS.2 antiserum were used to characterize hatching gland cells (HGCs) in birne shrimp embryos. The positive staining of HGC was first visible at the stage E2.1 (2 hours before hatching). As development of the embryos proceeds, the intensity of immunoreactivity and the number of positive HGCs increases and reaches the maximum at the stage of hatching, and decreased gradually after hatching. And the immunoreactivity signal gradually disappears 5 hours after hatching. The HGCs distributed all over the body of embryos 1 hour before hatching, while hatching, few
HGCs could be detected in the dorsal-anterior area of the embryos, even there were some heavily stained HGCs in the other dorsal area. The distribution of the brine shrimp HGCs varies greatly from the species studied till now. One hour after hatching, neither the dorsal-anterior area nor the other dorsal area remained positive immunoreactivity signal. And 2 hours after hatching, there was no typical HGCs in the body of the brine shrimp and the remained hatching enzymes may participate in digesting the left vitellin in the nauplius. From the above results it can be concluded that the dorsal-anterior area and the dorsal area are probably the main position of HE secretion.
免疫细胞化学染色结果显示，卤虫HGC最早出现于孵化前2h，于孵化后5h消失。卤虫HGC于孵化前2h刚刚出现时，胚胎中的HE颗粒界限不明显且数量较少，颜色也很淡，遍布于整个胚体；随胚胎的进一步发育，HGC的数量逐渐增多、颜色也逐渐变深，在孵化时达到最大，之后颗粒量便开始逐渐减少、颜色也逐渐变浅；孵化后2h的幼体中，HE阳性反应减弱，于胚胎局部已开始消失，且主要集中在胚胎中央及胚胎体表，最终于孵化后5h小时消失。卤虫HGC最初出现至孵化前1h时均为全身性分布，从孵出到孵出后2h，头
卤虫孵化酶的生物化学性质及孵化腺细胞的免疫组织化学研究
部的孵化酶颗粒已经减少，而变为非全身性分布，到孵出后sh，孵化酶颗粒
已基本消失殆尽。卤虫胚胎的头部很可能是孵化酶的最早开始分泌的部位，可
能也是主要的分泌部位，随着胚胎的继续孵出，身体背部继而腹部等部位也开
始分泌孵化酶，以协助胚胎完全孵出。孵出后仍残留的孵化酶有可能参与了卤
虫幼体内残余卵黄颗粒的消化降解，此结论还需进一步验证。
The hatching enzyme from brine shrimp, Artemia shrimp, is a pivotal protease which help the encysted embryo escape from its hatching membrane when hatching. The brine shrimp HE was prepared and purified by 67% ammonium sulfate precipitation, DEAE-sepharose Fast Flow ion-exchange chromatography, and Sephacryl column gel-filtration, and its biochemical and enzymological properties were identified in this study. It was found that the deduced molecular weight of HE in SDS-PAGE is about 98.5 kDa, and its proteolytic activity was optimized at pH of 7.5-8.5 and at temperature of 40 , respectively. The HE was strongly inhibited by SBTI and APMSF, and very sensitive to PMSF, LBTI, and TLCK, while not sensitive to chymostatin, bestatin, leupeptin,TPCK, pepstatin, NEM and IAM. All these results imply that brine shrimp HE was most probably a trypsin-type serine protease. The HE could be strongly inhibited by EDTA in a dose-dependent manner, and 50 mmol/L EDTA exhibited more than 56.5% inhibition. The brine shrimp HE w
as extremely sensitive to Zn2+, and 2.5 mmol/L Zn2+ could elevate HE activity for nearly 215 %, the HE was almost unaffected by Ca2+, Mg2+ and Cu2+. It can be concluded that the brine shrimp HE was propably a kind of Zn-metalloprotease.
The immunocytochemical staining methods employing anti-GST-UVS.2 antiserum were used to characterize hatching gland cells (HGCs) in birne shrimp embryos. The positive staining of HGC was first visible at the stage E2.1 (2 hours before hatching). As development of the embryos proceeds, the intensity of immunoreactivity and the number of positive HGCs increases and reaches the maximum at the stage of hatching, and decreased gradually after hatching. And the immunoreactivity signal gradually disappears 5 hours after hatching. The HGCs distributed all over the body of embryos 1 hour before hatching, while hatching, few
HGCs could be detected in the dorsal-anterior area of the embryos, even there were some heavily stained HGCs in the other dorsal area. The distribution of the brine shrimp HGCs varies greatly from the species studied till now. One hour after hatching, neither the dorsal-anterior area nor the other dorsal area remained positive immunoreactivity signal. And 2 hours after hatching, there was no typical HGCs in the body of the brine shrimp and the remained hatching enzymes may participate in digesting the left vitellin in the nauplius. From the above results it can be concluded that the dorsal-anterior area and the dorsal area are probably the main position of HE secretion.
引文
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