家蚕血淋巴中一种保幼激素结合蛋白的纯化与特性分析
Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm, Bombyx mori.
作者信息
Kurata K, Nakamura M, Okuda T, Hirano H, Shinbo H
机构信息
Department of Insect Physiology and Behavior, National Institute of Sericultural and Entomological Science, Ibaraki, Japan.
出版信息
Comp Biochem Physiol B Biochem Mol Biol. 1994 Sep;109(1):105-14. doi: 10.1016/0305-0491(94)90147-3.
A juvenile hormone binding protein (JHBP) has been isolated from Bombyx mori hemolymph by gel filtration, ion-exchange chromatography, chromatofocusing and hydroxyapatite column chromatography. Gel electrophoresis indicates that the isolated protein is homogeneous in the presence or absence of a denaturing agent. The JHBP in question has a relative molecular mass of 32 kDa, determined by denaturing gel electrophoresis. Chromatofocusing analysis indicated that the JHBP is an acidic protein with pI 4.9. The protein exhibits a dissociation constant of 9.0 x 10(-8) M for JH I, 1.14 x 10(-7) M for JH II and 3.9 x 10(-7) M for JH III, and thus its affinity for JH analogues is in the order of JH I > JH II > JH III. Its amino acid composition indicates that the protein consists of 297 residues of 18 kinds of amino acids. The sequence of the N-terminus of the polypeptide chain was determined for 34 of the first 36 residues: Asp-Gln-Asp-Ala-Leu-Leu- Lys-Pro-?-Lys-Leu-Gly-Asp-Met-Gln-Ser-Leu-Ser-Ser-Ala-Thr-Gln-Gln-Phe-Le u-Glu- Lys-Thr-Ser-Lys-Gly-Ile-Pro-?-Tyr-His-.
通过凝胶过滤、离子交换色谱、聚焦色谱和羟基磷灰石柱色谱法，从家蚕血淋巴中分离出一种保幼激素结合蛋白（JHBP）。凝胶电泳表明，在有或没有变性剂的情况下，分离出的蛋白质都是均一的。通过变性凝胶电泳测定，所讨论的JHBP相对分子质量为32 kDa。聚焦色谱分析表明，JHBP是一种酸性蛋白，其pI为4.9。该蛋白对保幼激素I（JH I）的解离常数为9.0×10⁻⁸ M，对保幼激素II（JH II）为1.14×10⁻⁷ M，对保幼激素III（JH III）为3.9×10⁻⁷ M，因此其对保幼激素类似物的亲和力顺序为JH I＞JH II＞JH III。其氨基酸组成表明该蛋白由18种氨基酸的297个残基组成。确定了多肽链N端前36个残基中34个残基的序列：天冬氨酸-谷氨酰胺-天冬氨酸-丙氨酸-亮氨酸-亮氨酸-赖氨酸-脯氨酸-？-赖氨酸-亮氨酸-甘氨酸-天冬氨酸-甲硫氨酸-谷氨酰胺-丝氨酸-亮氨酸-丝氨酸-丝氨酸-丙氨酸-苏氨酸-谷氨酰胺-谷氨酰胺-苯丙氨酸-亮氨酸-谷氨酸-赖氨酸-苏氨酸-丝氨酸-赖氨酸-甘氨酸-异亮氨酸-脯氨酸-？-酪氨酸-组氨酸-。
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