Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on activities of the transducin alpha subunit.
Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on activities of the transducin alpha subunit.
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牛转导蛋白的化学修饰：荧光素 5-异硫氰酸酯标记对转导蛋白 α 亚基活性的影响。
作者：
Hingorani,VN;Ho,Y
伊利诺伊大学芝加哥分校生物化学系，健康科学中心，芝加哥，伊利诺斯州60612摘要：荧光素S'-异硫氰酸酯（FITC）用于修饰牛转导蛋白(T)的赖氨酸残基，T是一种gtp结合蛋白，参与杆状光感受器细胞的光传导。FITC的掺入显示了大约1 mol FITC/mol转导素的化学计量。标记是针对Ta亚基的。在T^亚基上没有明显的合并。在紫红质存在的情况下，这种修饰对转导-视紫红质相互作用和鸟苷5/-（/i3，-y-咪唑三磷酸）[Gpp (NH) p]与转导蛋白的结合没有影响。fitc -转导素- gpp (NH) p复合物与视紫红质膜的分离保持不变。然而，经FITC修饰后，Ta的内在GTPase活性及其激活cGMP磷酸二酯酶的能力减弱。转导素- gpp (NH) p复合物的FITC标记率比转导素慢约3倍。有限的胰蛋白酶消化和肽定位用于定位FITC标记位点。FITC标签大部分位于23千道尔顿片段上，少量位于Ta亚基的9千道尔顿片段上。这些结果表明，FITC标记不会改变光解紫红质对转导蛋白的激活，但会影响GTP水解活性以及GTP诱导的Ta的构象变化，最终导致cGMP磷酸二酯酶的激活。
Department of Biological Chemistry, University of Illinois at Chicago, Health Sciences Center, Chicago, Illinois 60612 Received August 26, 1986; Revised Manuscript Received October 28, 1986 abstract: Fluorescein S'-isothiocyanate (FITC) was used to modify the lysine residues of bovine transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells. The incorporation of FITC showed a stoichiometry of approximately 1 mol of FITC/mol of transducin. The labeling was specific for the Ta subunit. There was no significant incorporation on the T^ subunit. The modification had no effect on the transducin-rhodopsin interaction or on the binding of guanosine 5/-(/i3,-y-imidotriphosphate)[Gpp (NH) p] to transducin in the presence of photolyzed rhodopsin. The dissociation of the FITC-transducin-Gpp (NH) p complex from rhodopsin membrane remained unchanged. However, the intrinsic GTPase activity of Ta and its ability to activate the cGMP phosphodiesterase were diminished by FITC modification. The rate of FITC labeling of the transducin-Gpp (NH) p complex was about 3-fold slower thanthat of transducin. Limited tryptic digestion and peptide mapping were used to localize the FITC labeling site. The majority of the FITC label was on the 23-kilodalton fragment, and a minor amount was on the 9-kilodalton fragment of the Ta subunit. These results indicatethat FITC labeling does not alter the activation of transducin by photolyzed rhodopsin but does affect the GTP hydrolytic activity as well as the GTP-induced conformational change of Ta, which ultimately leads to the activation of cGMP phosphodiesterase.