Studies On Properties Of Hatching Enzyme From Artemia Shrimp And Immunocytochemistry Of Hatching Gland Cells

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 was 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, fewHGCs 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.