Hamm H E, Deretic D, Hofmann K P, Schleicher A, Kohl B
J Biol Chem. 1987 Aug 5;262(22):10831-8.
Seven monoclonal antibodies to the alpha subunit (G alpha) of the frog photoreceptor guanyl nucleotide-binding protein (transducin or G-protein) have been characterized as to their effect on G-protein function, and this has been correlated in the accompanying paper (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) with the antibody-binding sites on G alpha tryptic fragments. Antibodies 4A, 7A, 7B, 7C, and 7D are members of a class of antibodies that block G-protein activation by light and therefore also block activation of the cGMP phosphodiesterase. All these blocking antibodies also block the interaction of G-protein with rhodopsin as measured by the light-scattering "binding signal," and as measured by the stabilization of meta-rhodopsin II by bound G-protein (extra-meta-rhodopsin II). The antibodies (or Fab fragments) also solubilize G alpha beta gamma from the membrane in the dark under isosmotic conditions and thus interfere with G alpha interaction with the membrane. Antibody 4A also blocks the extra-meta-rhodopsin II generated by G-protein-rhodopsin interaction in detergent solubilized membranes. Thus, even in the absence of phospholipids, antibody 4A blocks G-protein-rhodopsin interaction. Therefore, we suggest that the antibodies recognize a region of G alpha involved with binding to rhodopsin. An alternative hypothesis is that this antigenic site is a region of interaction between the alpha and beta gamma subunits, disruption of this interaction leading to removal of both the alpha and beta gamma subunits from the membrane and blocking interaction with rhodopsin. This does not seem to be the case because the antibodies immunoprecipitate the alpha beta gamma complex, and not just the alpha subunit. Other antibodies, 4C and 4H, do not block phosphodiesterase activation, the light-scattering signal, extra-meta-rhodopsin II formation, or interaction with the membrane in the dark and therefore recognize other sites on G alpha.
已对七种针对蛙类光感受器鸟苷酸结合蛋白（转导素或G蛋白）α亚基（Gα）的单克隆抗体的G蛋白功能效应进行了表征，并且在随附论文（Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839 - 10847）中，将其与Gα胰蛋白酶片段上的抗体结合位点相关联。抗体4A、7A、7B、7C和7D属于一类抗体，这类抗体可通过光阻断G蛋白激活，因此也可阻断cGMP磷酸二酯酶的激活。所有这些阻断性抗体还可通过光散射“结合信号”以及通过结合的G蛋白（额外的间视紫红质II）对间视紫红质II的稳定作用来阻断G蛋白与视紫红质的相互作用。这些抗体（或Fab片段）在等渗条件下的黑暗中也能使Gαβγ从膜中溶解，从而干扰Gα与膜的相互作用。抗体4A还可阻断去污剂溶解膜中由G蛋白 - 视紫红质相互作用产生的额外间视紫红质II。因此，即使在没有磷脂的情况下，抗体4A也能阻断G蛋白 - 视紫红质相互作用。所以，我们认为这些抗体识别的是Gα中与视紫红质结合相关的区域。另一种假说是，这个抗原位点是α亚基和βγ亚基之间的相互作用区域，这种相互作用的破坏导致α亚基和βγ亚基都从膜上脱离，并阻断与视紫红质的相互作用。但情况似乎并非如此，因为这些抗体免疫沉淀的是αβγ复合物，而不仅仅是α亚基。其他抗体4C和4H不会阻断磷酸二酯酶激活、光散射信号、额外间视紫红质II的形成或在黑暗中与膜的相互作用，因此识别的是Gα上的其他位点。
