Purification and Properties of Two Types of Soluble Trehalases from Embryonic Larvae of the Silkworm, Bombyx mori
Purification and Properties of Two Types of Soluble Trehalases from Embryonic Larvae of the Silkworm, Bombyx mori
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DOI：
10.11416/jibs.75.1
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发表时间：
2006-02
影响因子：
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通讯作者：
Junyi Huang;T. Furusawa;Keiko Sadakane;Y. Sugimura
中科院分区：
文献类型：
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作者：
Junyi Huang;T. Furusawa;Keiko Sadakane;Y. Sugimura
为了确定家蚕发育卵中海藻糖酶的性质，采用酸处理、sepphacryl S-300、deae -纤维素和Con A-Sepharose柱层析和制备聚丙烯酰胺凝胶电泳等方法，从家蚕卵匀浆上清中纯化出可溶性型海藻糖酶。凝胶过滤柱层析法检测到两个海藻酶活性峰，分别为PI （73 kDa）和P II （140 kDa）。在NaCl浓度为0.2 M和0.25 M时，通过离子交换柱层析得到PI和PI为单峰。经SDS-PAGE和Western blot分析，PI和P II的亚基分子质量约为64 kDa。海藻糖的PI的Km为1.56 mM，最佳pH为6.5左右，活化能为11.03 kCal/mol。Pⅱ对海藻糖的Km为0.44 mM，最适pH为5.5左右，活化能为5.92 kCal/mol，明显较低。这些结果表明，在家蚕的胚胎幼虫中存在两种类型的可溶性海藻糖酶。
In order to determine the properties of trehalase in the developing eggs of Bombyx mori, soluble-type trehalase was purified from the supernatant of egg homogenate by acid treatment, column chromatographies of Sephacryl S-300, DEAE-cellulofine and Con A-Sepharose, and preparative polyacrylamide gel electrophoresis. Two peaks of trehalase activity were detected by gel filtration column chromatography, and designated as PI (73 kDa) and P II (140 kDa). PI and P II were eluted as a single peak from ion exchange column chromatography at about 0.2 M and 0.25 M of NaCl, respectively. Subunit molecular masses of PI and P II were estimated as approximately 64 kDa on SDS-PAGE and Western blot analysis. PI had a Km of 1.56 mM for trehalose and an optimal pH around 6.5 with an activation energy of 11.03 kCal/mol. P II had a Km of 0.44 mM for trehalose and an optimal pH around 5.5 with an apparently lower activation energy of 5.92 kCal/mol. These results suggest that two types of soluble trehalase are present in the embryonic larvae of the silkworm, Bombyx mori.