Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm, Bombyx mori

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry Pub Date : 1994-09-01 DOI:10.1016/0305-0491(94)90147-3

Keiji Kurata , Masatoshi Nakamura , Takashi Okuda , Hisashi Hirano , Hiroshi Shinbo

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引用次数: 18

Abstract

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 × 10⁻⁸ M for JH I, 1.14 × 10⁻⁷ M for JH II and 3.9 × 10⁻⁷ M for JH III, and thus its affinity for JH analogues is in the order of JHI >JHII >JHIII. 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-Leu-Glu- Lys-Thr-Ser-Lys-Gly-Ile-Pro-?-Tyr-His-.

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家蚕血淋巴幼体激素结合蛋白的纯化及特性研究

采用凝胶过滤、离子交换层析、聚焦层析和羟基磷灰石柱层析等方法，从家蚕血淋巴中分离到一种幼年激素结合蛋白(JHBP)。凝胶电泳表明，分离的蛋白质在存在或不存在变性剂的情况下是均匀的。通过变性凝胶电泳测定，JHBP的相对分子质量为32 kDa。染色质聚焦分析表明，JHBP为酸性蛋白，pI为4.9。该蛋白对JHI的解离常数为9.0 × 10−8 M，对JHII的解离常数为1.14 × 10−7 M，对JHIII的解离常数为3.9 × 10−7 M，因此其对JH类似物的亲和力为JHI >JHII >JHIII。其氨基酸组成表明，该蛋白由18种氨基酸的297个残基组成。确定了前36个残基中34个多肽链n端序列:Asp-Gln-Asp-Ala-Leu-Leu-Lys-Pro-?-Lys-Leu-Gly-Asp-Met-Gln-Ser-Leu-Ser-Ser-Ala-Thr-Gln-Gln-Phe-Leu-Glu - Lys-Thr-Ser-Lys-Gly-Ile-Pro ? -Tyr-His -。

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Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

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