Limits...
Characterization of a novel hatching enzyme purified from starfish Asterina pectinifera

View Article: PubMed Central - PubMed

ABSTRACT

Hatching enzyme is a protease which can degrade the membrane of egg. In this study, a hatching enzyme was purified from starfish (Asterina pectinifera) with 6.34 fold of purification rate, 5.04 % of yield, and 73.87 U/mg of specific activity. The molecular weight of starfish hatching enzyme was 86 kDa, which was reduced to 62 kDa after removal of N-linked oligosaccharides. The optimal pH and temperature of the hatching enzyme activity were pH 7.0 and 40 °C, respectively, while those of stability were pH 8 and 20 °C. The kinetic parameters, Vmax, Km, Kcat and Kcat/Km values were 0.197 U/ml, 0.289 mg/ml, 112.57 s−1, and 389.52 ml/mg s, respectively. Zn2+ increased the enzyme activity by 167.28 %, while EDTA, TPCK, TGCK, leupeptin, PMSF, and TLCK decreased. In addition, Ca2+, Mg2+, and Cu2+ did not affect the enzyme activity. The starfish hatching enzyme activity pretreated with EDTA was recovered by Zn2+. Therefore, the starfish hatching enzyme was classified as a serine-zinc protease.

No MeSH data available.


Elution profile of the starfish hatching enzyme. a DEAE-Ion exchange chromatography. b Sephachryl gel filtration chromatography
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120168&req=5

Fig1: Elution profile of the starfish hatching enzyme. a DEAE-Ion exchange chromatography. b Sephachryl gel filtration chromatography

Mentions: Hatching enzyme of starfish was purified using an ammonium sulfate precipitation, DEAE-Sepharose ion exchange and Sephacryl S-200 gel filtration column chromatograpy, in that order. DEAE-Sepharose ion exchange column chromatography resulted in two protein peaks with choriolytic activity (Fig. 1a). The yield, specific choriolytic activity, and purification ratio of peak I and II were 33.37 and 46.28 %, 30.22 and 23.76 U/mg, and 2.59 and 2.04 fold, respectively (Table 1). Because of higher specific choriolytic activity, the peak I was further purified using a Sephacryl S-200 column chromatography, which resulted in only one protein peak (Fig. 1b). The purification rate, yield, and specific choriolytic activity of the purified hatching enzyme were 6.34 fold, 5.04 %, and 73.87 U/mg, respectively (Table 1). The hatching enzyme with molecular weight of 86 kDa was homogeneous on the SDS-PAGE (Fig. 2a). The specific choriolytic activity (73.87 U/mg) of the starfish hatching enzyme in this study was lower than 400.00 U/mg of brine shrimp (Fan et al. 2010) and 449.62 U/mg of starfish A. amurensis (Li and Kim 2013). The purification rate and yield of starfish hatching enzyme (6.34 fold and 5.04 %) in this study were also lower than those of starfish A. amurensis (7.42 fold and 14.28 %) (Li and Kim 2013), shrimp (48.05 fold and 44.29 %) (Li et al. 2006), sea urchin (201 fold and 53 %) (Roe and Lennarz 1990), and sea squirt (67.8 fold and 29.4 %) (D’Aniello et al. 1997). These differences might be due to different species, preparations, and purification methods. The molecular weight of the starfish hatching enzyme in this study was 86 kDa, which was a smaller than 110.9 kDa of A. amurensis (Li and Kim 2013), but a little higher than 73.3 kDa of brine shrimp (Fan et al. 2010). However, it was much higher than those of the hatching enzyme from shrimp (43 kDa) (Li et al. 2006), sea urchin (37, 44, 51 kDa) (Lepage and Gache 1989; Nomura et al. 1991; Takeuchi et al. 1979), frog (40, 56 kDa) (Fan and Katagiri 2001; Kitamura and Katagiri 1998), sea squirt (34 kDa) (D’Aniello et al. 1997), flounder (34.8 kDa) (Shi et al. 2010), Fundulus heteroclitus (15–40 kDa) (DiMichele et al. 1981), Oryzias latipes (LCE 25.5 kDa; HCE 24 kDa) (Yasumasu et al. 1989a, b), and Salmo gairdneri (10 kDa) (Hagenmaier 1974). The PNGase F was used to release the asparagine-linked (N-linked) oligosaccharides from the hatching enzyme protein. After PNGase F treatment, the band of hatching enzyme protein with 86 kDa was shifted to 62 kDa (Fig. 2b). Therefore, the 24 kDa of N-linked oligosaccharides was removed from the hatching enzyme protein. The molecular weight of the starfish hatching enzyme, 86 kDa, was quietly different from those of other animals (shrimp, sea urchin, frog, sea squirt, flounder, mummichog, medaka, and rainbow trout) including 110.9 kDa of starfish Asterias amurensis. In addition, Tbrain or T-box brain protein 1 is a transcription factor protein important in vertebrate embryo development. It is encoded by the TBR1 gene which is involved in the mesoderm formation of vertebrate embryos. Mammalian T-brain is expressed in the developing central nervous system. Hinman et al. (2007) reported the results of gene analysis of sea stars and sea urchins as follows; it has been conserved for 500 million years since sea stars and sea urchins last shared a common ancestor. Amid this high level of conservation, one significant regulatory change was elucidated. Tbrain was required for correct otxβ1/2 expression in the sea star, but not in the sea urchin. In sea urchin, Tbrain was not co-expressed with otxβ1/2 and instead had an essential role in specification of the embryonic skeleton. Tbrain in these echinoderms was thus a perfect example of an orthologous gene co-opted for entirely different developmental processes. According to above explanations, the starfish hatching enzyme might be ortholog.Fig. 1


Characterization of a novel hatching enzyme purified from starfish Asterina pectinifera
Elution profile of the starfish hatching enzyme. a DEAE-Ion exchange chromatography. b Sephachryl gel filtration chromatography
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120168&req=5

Fig1: Elution profile of the starfish hatching enzyme. a DEAE-Ion exchange chromatography. b Sephachryl gel filtration chromatography
Mentions: Hatching enzyme of starfish was purified using an ammonium sulfate precipitation, DEAE-Sepharose ion exchange and Sephacryl S-200 gel filtration column chromatograpy, in that order. DEAE-Sepharose ion exchange column chromatography resulted in two protein peaks with choriolytic activity (Fig. 1a). The yield, specific choriolytic activity, and purification ratio of peak I and II were 33.37 and 46.28 %, 30.22 and 23.76 U/mg, and 2.59 and 2.04 fold, respectively (Table 1). Because of higher specific choriolytic activity, the peak I was further purified using a Sephacryl S-200 column chromatography, which resulted in only one protein peak (Fig. 1b). The purification rate, yield, and specific choriolytic activity of the purified hatching enzyme were 6.34 fold, 5.04 %, and 73.87 U/mg, respectively (Table 1). The hatching enzyme with molecular weight of 86 kDa was homogeneous on the SDS-PAGE (Fig. 2a). The specific choriolytic activity (73.87 U/mg) of the starfish hatching enzyme in this study was lower than 400.00 U/mg of brine shrimp (Fan et al. 2010) and 449.62 U/mg of starfish A. amurensis (Li and Kim 2013). The purification rate and yield of starfish hatching enzyme (6.34 fold and 5.04 %) in this study were also lower than those of starfish A. amurensis (7.42 fold and 14.28 %) (Li and Kim 2013), shrimp (48.05 fold and 44.29 %) (Li et al. 2006), sea urchin (201 fold and 53 %) (Roe and Lennarz 1990), and sea squirt (67.8 fold and 29.4 %) (D’Aniello et al. 1997). These differences might be due to different species, preparations, and purification methods. The molecular weight of the starfish hatching enzyme in this study was 86 kDa, which was a smaller than 110.9 kDa of A. amurensis (Li and Kim 2013), but a little higher than 73.3 kDa of brine shrimp (Fan et al. 2010). However, it was much higher than those of the hatching enzyme from shrimp (43 kDa) (Li et al. 2006), sea urchin (37, 44, 51 kDa) (Lepage and Gache 1989; Nomura et al. 1991; Takeuchi et al. 1979), frog (40, 56 kDa) (Fan and Katagiri 2001; Kitamura and Katagiri 1998), sea squirt (34 kDa) (D’Aniello et al. 1997), flounder (34.8 kDa) (Shi et al. 2010), Fundulus heteroclitus (15–40 kDa) (DiMichele et al. 1981), Oryzias latipes (LCE 25.5 kDa; HCE 24 kDa) (Yasumasu et al. 1989a, b), and Salmo gairdneri (10 kDa) (Hagenmaier 1974). The PNGase F was used to release the asparagine-linked (N-linked) oligosaccharides from the hatching enzyme protein. After PNGase F treatment, the band of hatching enzyme protein with 86 kDa was shifted to 62 kDa (Fig. 2b). Therefore, the 24 kDa of N-linked oligosaccharides was removed from the hatching enzyme protein. The molecular weight of the starfish hatching enzyme, 86 kDa, was quietly different from those of other animals (shrimp, sea urchin, frog, sea squirt, flounder, mummichog, medaka, and rainbow trout) including 110.9 kDa of starfish Asterias amurensis. In addition, Tbrain or T-box brain protein 1 is a transcription factor protein important in vertebrate embryo development. It is encoded by the TBR1 gene which is involved in the mesoderm formation of vertebrate embryos. Mammalian T-brain is expressed in the developing central nervous system. Hinman et al. (2007) reported the results of gene analysis of sea stars and sea urchins as follows; it has been conserved for 500 million years since sea stars and sea urchins last shared a common ancestor. Amid this high level of conservation, one significant regulatory change was elucidated. Tbrain was required for correct otxβ1/2 expression in the sea star, but not in the sea urchin. In sea urchin, Tbrain was not co-expressed with otxβ1/2 and instead had an essential role in specification of the embryonic skeleton. Tbrain in these echinoderms was thus a perfect example of an orthologous gene co-opted for entirely different developmental processes. According to above explanations, the starfish hatching enzyme might be ortholog.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Hatching enzyme is a protease which can degrade the membrane of egg. In this study, a hatching enzyme was purified from starfish (Asterina pectinifera) with 6.34 fold of purification rate, 5.04 % of yield, and 73.87 U/mg of specific activity. The molecular weight of starfish hatching enzyme was 86 kDa, which was reduced to 62 kDa after removal of N-linked oligosaccharides. The optimal pH and temperature of the hatching enzyme activity were pH 7.0 and 40 °C, respectively, while those of stability were pH 8 and 20 °C. The kinetic parameters, Vmax, Km, Kcat and Kcat/Km values were 0.197 U/ml, 0.289 mg/ml, 112.57 s−1, and 389.52 ml/mg s, respectively. Zn2+ increased the enzyme activity by 167.28 %, while EDTA, TPCK, TGCK, leupeptin, PMSF, and TLCK decreased. In addition, Ca2+, Mg2+, and Cu2+ did not affect the enzyme activity. The starfish hatching enzyme activity pretreated with EDTA was recovered by Zn2+. Therefore, the starfish hatching enzyme was classified as a serine-zinc protease.

No MeSH data available.