Zamudio F Z, Conde R, Arévalo C, Becerril B, Martin B M, Valdivia H H, Possani L D
Department of Molecular Recognition and Structural Biology, Biotechnology Institute, National Autonomous University of Mexico, Cuernavaca, Morelos 62271, Mexico.
J Biol Chem. 1997 May 2;272(18):11886-94. doi: 10.1074/jbc.272.18.11886.
We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an approximately 15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta-bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta-bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.
我们对帝王毒素I（IpTxi）的结构和功能特性进行了深入分析。IpTxi是一种来自帝王蝎毒液的约15 kDa蛋白质，可抑制Ca2+释放通道/兰尼碱受体（RyR）的活性（瓦尔迪维亚，H. H.，柯比，M. S.，莱德勒，W. J.，和科罗纳多，R.（1992年）《美国国家科学院院刊》89，12185 - 12189）。从这种蝎子的毒腺制备了一个cDNA文库，并用于克隆编码IpTxi的基因。从单个连续的信使RNA中，编码毒素的信息在去除一个碱性五肽后被翻译成两个成熟的多肽亚基。IpTxi二聚体由一个具有磷脂酶A2（PLA2）活性的大亚基（104个氨基酸残基）通过二硫键与一个较小的（27个氨基酸残基）、结构不相关的亚基共价连接而成。因此，IpTxi是一种具有脂解作用的异源二聚体蛋白，这一特性仅与β - 银环蛇毒素（一组来自蛇毒的神经毒素）相同。IpTxi的酶亚基与来自蜜蜂（意大利蜜蜂）和蜥蜴（珠毒蜥）毒液的PLA2高度同源。小亚基与任何其他已知肽没有显著相似性，包括针对β - 银环蛇毒素脂解作用的库尼茨蛋白酶抑制剂超家族成员。一个氨基酸序列与小亚基相同的合成肽未能抑制RyR。另一方面，用对溴苯甲酰溴（一种PLA2活性的特异性抑制剂）处理IpTxi，大大降低了IpTxi抑制RyR的能力。这些结果表明，PLA2活性的脂质产物而非直接的IpTxi - RyR相互作用是导致RyR抑制的原因。
