Abramson S N, Culver P, Kline T, Li Y, Guest P, Gutman L, Taylor P
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla 92093.
J Biol Chem. 1988 Dec 5;263(34):18568-73.
Lophotoxin and lophotoxin analog-1 are uncharged cyclic diterpenes obtained from gorgonian corals. They have been shown to block the function of nicotinic acetylcholine receptors. Inhibition results from blockade of the agonist recognition site and appears irreversible in that extensive washing does not restore receptor function. This study was undertaken to determine whether this apparently irreversible inhibition involves covalent labeling at a selective site and to further characterize this site directly. Incubation of membranes prepared from the electric organ of Torpedo californica with analog-1 followed by reduction with NaB3H4 resulted in the incorporation of radioactivity into several membrane proteins. The incorporation of radioactivity into the alpha-subunit of the receptor was blocked by prior incubation with agonists and antagonists. [3H]Lophotoxin and [3H]analog-1 were prepared by reduction with NaB3H4 and back-oxidation with CrO3. The radiolabeled coral toxins reacted selectively and covalently with the alpha-subunit of the receptor. Their binding was prevented by prior exposure to agonists and antagonists. In contrast to the site-directed alkylating agent 4-(N-maleimido)benzyltrimethylammonium iodide, prior reduction of the receptor was not required for covalent binding of 3H-labeled coral toxins. Selective reduction of Cys192 and Cys193 followed by alkylation with 4-(N-maleimido)benzyltrimethylammonium iodide blocked the binding of [3H]analog-1, whereas alkylation with iodoacetic acid or iodoacetamide did not. Thus, the binding site for the coral toxins does not overlap the binding surface near Cys192 and Cys193. Digestion of isolated labeled alpha-subunits with endoglycosidase H revealed that the polypeptide portion of the protein retained the covalently bound [3H]analog-1. Digestion with staphylococcal V8 protease revealed two major peptides of approximately 19 and 20 kDa, along with several smaller peptides. Only the 20-kDa peptide retained the covalently bound [3H]analog-1, localizing the site of covalent attachment between Ser173 and Glu335. The unique chemical structure and covalent reactivity of these gorgonian coral toxins will undoubtedly allow further insights into the structure of the agonist recognition site.
洛伐他汀和洛伐他汀类似物 -1是从柳珊瑚中提取的不带电荷的环状二萜类化合物。它们已被证明可阻断烟碱型乙酰胆碱受体的功能。抑制作用源于对激动剂识别位点的阻断，并且由于大量洗涤不能恢复受体功能，这种抑制作用似乎是不可逆的。本研究旨在确定这种明显不可逆的抑制作用是否涉及在选择性位点的共价标记，并直接进一步表征该位点。用类似物 -1孵育加州电鳐电器官制备的膜，然后用NaB3H4还原，导致放射性掺入几种膜蛋白中。放射性掺入受体的α亚基被预先与激动剂和拮抗剂孵育所阻断。[3H]洛伐他汀和[3H]类似物 -1通过用NaB3H4还原和用CrO3反氧化制备。放射性标记的珊瑚毒素与受体的α亚基选择性地共价反应。它们的结合被预先暴露于激动剂和拮抗剂所阻止。与位点定向烷基化剂4-(N-马来酰亚胺基)苄基三甲基碘化铵相反，3H标记的珊瑚毒素的共价结合不需要预先还原受体。选择性还原Cys192和Cys193，然后用4-(N-马来酰亚胺基)苄基三甲基碘化铵烷基化，阻断了[3H]类似物 -1的结合，而用碘乙酸或碘乙酰胺烷基化则没有。因此，珊瑚毒素的结合位点与Cys192和Cys193附近的结合表面不重叠。用内切糖苷酶H消化分离的标记α亚基表明，蛋白质的多肽部分保留了共价结合的[3H]类似物 -1。用葡萄球菌V8蛋白酶消化显示出两条主要的肽段，大小约为19 kDa和20 kDa，以及几条较小的肽段。只有20 kDa的肽段保留了共价结合的[3H]类似物 -1，将共价连接位点定位在Ser173和Glu335之间。这些柳珊瑚毒素独特的化学结构和共价反应性无疑将有助于进一步深入了解激动剂识别位点的结构。
