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甘味タンパク質の甘味活性発現機構

甜味蛋白甜味活性表达机制

基本信息

批准号：
08660156
负责人：
KITABATAKE Naofumi
金额：
$ 1.47万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

Thaumatin, a sweet protein that contains no cysteine residues and eight intramolecular disulfide bonds, aggregates upon heating at pH 7.0 above 70℃ and its sweetness thereby disappears. The aggregate can be solubilized by heating in the presence of both thiol reducjng reagent and SDS. This molecular aggregation depended on the protein concentration during heating and was suppressed by the addition of N-ethylmaleimide or iodoacetamide, indicating a thiol-catalyzed disulfide interchange reaction between heat-denatured molecules. An amino acid analysis of the aggregates suggested that the cysteine and lysine residues were reduced, and the formation of a cysteine residue and a lysinoalanine residue was confirmed. The reduction and formation of these residues stoichiometrically satisfied the B-elimination of a cystine residue. The disulfide interchange reaction was catalyzed by cysteine ; I.e., a free sulfhydryl residue was formed via B-elimination of a disulfide bond. Intermolecular disulf … More ide bond were probably formed between thaumatin molecules upon heating at pH 7.0, which led to the aggregation of thaumatin molecules.Thaumatin I is the sweet tasting protein isolated from the arils of a plant native to tropical West Africa. However, despite its strong sweetness, the structural basis for its sweetness is still unknown. We identified the functionally important lysine residues by using chemical modification study. Pyridoxal 5'-phosphate (PLP) was used to selectively modify lysine residues on thaumatin I. PLP was incubated with thaumatin I at pH 7.0, and the schiff base was reduced with sodium borohydride. Five modified thaumatin I, all of which were incorporated one PLP per thaumatin I molecule, were purified and its modified lysine residues were identified. Lys78, kys97, Lys106, Lys137, and Lys187 were modified, and any of these modification did not affect the conformation of thaumatin I as inferred from the measurement of circular dichroism spectra. Modified thaumatin I showed reduced sweetness, 67-85% loss. The modified thaumatin I was treated with phosphatase to remove phosphate group of attached PLP. Removal from Lys78-, Lys97-, Lys137-, and Lys187-modified-thaumatin I restored intense sweetness comparable to that of native thaumatin I, whereas removal from Lys106-modified-thaumatin I failed to restore sweetness. These results reveal that these five lysine residues, Lys78, Lys97, Lys106, Lys137, and Lys187 are functionally important. Our results also suggest, although the roles of Lys106 is obscure, that positive charge of Lys78, Lys97, Lys137, and Lys187 is participated in electrostatic interaction with the putative thaumatin's receptor. Less

奇异果甜蛋白是一种不含半胱氨酸残基和8个分子内二硫键的甜味蛋白，在pH 7.0高于70℃加热时聚集，其甜味因此消失。聚集体可以通过在硫醇还原剂和SDS两者的存在下加热来溶解。这种分子聚集依赖于加热过程中的蛋白质浓度，并通过添加N-乙基马来酰亚胺或碘乙酰胺抑制，表明热变性分子之间的硫醇催化的二硫键交换反应。聚集体的氨基酸分析表明，半胱氨酸和赖氨酸残基被还原，并证实了半胱氨酸残基和赖氨酸丙氨酸残基的形成。这些残基的还原和形成在化学计量上满足胱氨酸残基的B-消除。二硫键交换反应由半胱氨酸催化;即，通过二硫键的B-消除形成游离巯基残基。分子间二硫 ...更多信息 Thaumatin I是从西非热带植物的假种皮中分离得到的一种甜味蛋白。然而，尽管它的甜味很强，但其甜味的结构基础仍然未知。我们通过化学修饰研究确定了功能上重要的赖氨酸残基。吡哆醛5 '-磷酸（PLP）用于选择性修饰索马甜蛋白I上的赖氨酸残基。将PLP与奇异果甜蛋白I在pH 7.0下孵育，并用硼氢化钠还原席夫碱。纯化了5个修饰的索马甜I，每个索马甜I分子都掺入了一个PLP，并鉴定了其修饰的赖氨酸残基。Lys 78、kys 97、Lys 106、Lys 137和Lys 187被修饰，并且这些修饰中的任何一个都不影响索马甜I的构象，如从圆二色光谱的测量所推断的。修饰的索马甜I显示出降低的甜度，损失67-85%。用磷酸酶处理修饰的索马甜I以去除附着的PLP的磷酸基团。从Lys 78-、Lys 97-、Lys 137-和Lys 187-修饰的索马甜I中去除后，恢复了与天然索马甜I相当的强烈甜味，而从Lys 106-修饰的索马甜I中去除后，未能恢复甜味。这些结果表明，这五个赖氨酸残基，Lys 78，Lys 97，Lys 106，Lys 137和Lys 187是功能上重要的。我们的研究结果还表明，虽然Lys 106的作用是模糊的，Lys 78，Lys 97，Lys 137和Lys 187的正电荷参与静电相互作用与假定的索马甜受体。少