Aloj S M, Lee G, Consiglio E, Formisano S, Minton A P, Kohn L D
J Biol Chem. 1979 Sep 25;254(18):9030-9.
A strongly fluorescent 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of bovine thyrotropin has been prepared. The dye-conjugated hormone is bioactive and shares, essentially unchanged, the membrane binding and adenylate cyclase stimulatory activities of the native hormone. Binding of 125I-labeled dansyl-thyrotropin to thyroid plasma membranes is sensitive to inhibition by gangliosides and, as is the case for the binding of 125I-thyrotropin, galactosyl-N-acetylgalactosaminyl[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GDIb) is the most potent binding inhibitor. Gangliosides interact with dansyl-thyrotropin, causing a large increase of the quantum yield and a 5- to 10-nm blue shift of the emission maximum of the hormone-bound naphthalene chromophore; gangliosides cause no change in the fluorescent properties of the free dye. The fluorescence enhancement caused by gangliosides can be specifically reversed by unlabeled thyrotropin. The effect of gangliosides on dansyl-thyrotropin fluorescence is strongly salt-dependent; salts cannot, however, reverse the formation of the dansyl-thyrotropin.ganglioside complex once it has formed. The salt data suggest that the association of the ganglioside with dansyl-thyrotropin is dominated by electrostatic interactions, but that salt-independent, short range interactions, most likely hydrophobic, dominate the dissociation of the dansyl-thyrotropin-ganglioside adduct. Sucrose gradient centrifugation, ultracentrifugation, and fluorescence polarization data indicate that the gangliosides are micellar in nature under the conditions of these experiments. Acid titration of dansyl-thyrotropin causes a marked quenching of dansyl fluorescence which in part reflects dissociation of the hormone into its constituent alpha and beta subunits. In the presence of GDIb, but not N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GDIa), pH-dependent quenching and subunit dissociation are essentially eliminated. Circular dichroism results and fluorescence polarization studies support the interpretation that the ganglioside interaction causes a conformational change in the thyrotropin molecule. The acid titration data together with differences in the ability of gangliosides to influence the tyrosine fluorescence of the thyrotropin molecule indicate that different gangliosides induce different conformational perturbations in the thyrotropin molecule.
已制备出一种牛促甲状腺激素的强荧光5-二甲基氨基-1-萘磺酸盐（丹磺酰）衍生物。这种染料偶联激素具有生物活性，并且基本上保留了天然激素的膜结合和腺苷酸环化酶刺激活性。125I标记的丹磺酰促甲状腺激素与甲状腺质膜的结合对神经节苷脂的抑制敏感，并且与125I-促甲状腺激素的结合情况一样，半乳糖基-N-乙酰半乳糖胺基[双（N-乙酰神经氨酸）]-半乳糖基葡糖神经酰胺（GD1b）是最有效的结合抑制剂。神经节苷脂与丹磺酰促甲状腺激素相互作用，导致量子产率大幅增加，并且激素结合的萘发色团的发射最大值发生5至10纳米的蓝移；神经节苷脂对游离染料的荧光特性没有影响。神经节苷脂引起的荧光增强可被未标记的促甲状腺激素特异性逆转。神经节苷脂对丹磺酰促甲状腺激素荧光的影响强烈依赖于盐；然而，一旦丹磺酰促甲状腺激素-神经节苷脂复合物形成，盐就无法使其解离。盐的数据表明，神经节苷脂与丹磺酰促甲状腺激素的结合主要由静电相互作用主导，但盐不依赖的短程相互作用，很可能是疏水相互作用，主导了丹磺酰促甲状腺激素-神经节苷脂加合物的解离。蔗糖梯度离心、超速离心和荧光偏振数据表明，在这些实验条件下，神经节苷脂本质上是胶束状的。丹磺酰促甲状腺激素的酸滴定导致丹磺酰荧光显著猝灭，这部分反映了激素解离成其组成的α和β亚基。在存在GD1b但不存在N-乙酰神经氨酸半乳糖基-N-乙酰半乳糖胺基[单（N-乙酰神经氨酸）]-半乳糖基葡糖神经酰胺（GD1a）的情况下，pH依赖性猝灭和亚基解离基本消除。圆二色性结果和荧光偏振研究支持这样的解释，即神经节苷脂相互作用导致促甲状腺激素分子发生构象变化。酸滴定数据以及神经节苷脂影响促甲状腺激素分子酪氨酸荧光能力的差异表明，不同的神经节苷脂在促甲状腺激素分子中诱导不同的构象扰动。
