Fulachier M H, Mourier G, Cotton J, Servent D, Ménez A
Département d'Ingénierie et d'Etudes des Protéines, C.E.A., Saclay, Gif/Yvette, France.
FEBS Lett. 1994 Feb 7;338(3):331-8. doi: 10.1016/0014-5793(94)80294-7.
Using a solid-phase assay, we found that 3H-labeled alpha Cobtx from Naja naja siamensis, a long-chain curaremimetic toxin, and 3H-labelled toxin alpha from Naja nigricollis, a short-chain toxin both bind specifically but with substantially different affinities (Kd = 4 x 10(-7) M and 50 x 10(-6) M) to fragment 185-199 (T alpha 185-199) of the alpha-subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long-chain and short-chain toxins (Tyr-25, Lys-27, Trp-29, and Lys-53) or to long-chain toxins only (Cys-30 and Cys-34) do not affect the binding of the toxins to T alpha 185-199, suggesting that none of these invariant residues in implicated in the recognition of this AcChoR region. alpha Cobtx and toxin alpha bind to the fragment 128-142 (T alpha 128-142) with more similar affinities (Kd = 3 x 10(-7) M and 1.4 x 10(-6) M) and their binding is dramatically affected by the single abolition of the positive charge of Lys-53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys-53 more specifically recognizes the 128-142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.
使用固相分析方法，我们发现，来自眼镜蛇（Naja naja siamensis）的3H标记的α-椰毒（一种长链箭毒样毒素）和来自黑颈眼镜蛇（Naja nigricollis）的3H标记的毒素α（一种短链毒素）均能特异性结合，但与电鳐（Torpedo marmorata）乙酰胆碱受体（AcChoR）α亚基的185 - 199片段（Tα185 - 199）的亲和力存在显著差异（解离常数Kd分别为4×10⁻⁷M和50×10⁻⁶M）。然后我们发现，长链和短链毒素共有的残基（酪氨酸 - 25、赖氨酸 - 27、色氨酸 - 29和赖氨酸 - 53）或仅长链毒素特有的残基（半胱氨酸 - 30和半胱氨酸 - 34）的单衍生化并不影响毒素与Tα185 - 199的结合，这表明这些不变残基均不参与对该AcChoR区域的识别。α-椰毒和毒素α与128 - 142片段（Tα128 - 142）的结合亲和力更为相似（Kd分别为3×10⁻⁷M和1.4×10⁻⁶M），并且它们的结合会因赖氨酸 - 53（一个有助于AcChoR识别的不变残基）正电荷的单一消除而受到显著影响。因此，数据表明赖氨酸 - 53更特异性地识别AcChoR的128 - 142区域。其他单衍生化对毒素结合没有影响。本文所述方法可能对鉴定与受体片段直接接触的毒素残基有很大帮助。
