Wong S K, Martin B R, Tolkovsky A M
Biochem J. 1985 Nov 15;232(1):191-7. doi: 10.1042/bj2320191.
We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.
我们比较了鸟嘌呤核苷酸和Mg2+对百日咳博德特氏菌毒素（IAP）和霍乱毒素（CT）催化的大鼠脑和肝膜蛋白ADP核糖基化的影响。在[α-32P]NAD+、ATP和CT存在的情况下，蛋白质标记需要GTP或鸟苷5'-[γ-硫代]三磷酸（GTP[S]）。相比之下，IAP对一种（肝脏）或两种（脑）多肽的标记可被鸟苷5'-[β-硫代]二磷酸（GDP[S]）或GTP增强，但被GTP[S]或鸟苷5'-[β,γ-亚氨基]三磷酸（p[NH]ppG）阻断。标记强度顺序为GDP[S]＞GTP＞未添加＞GTP[S]=p[NH]ppG。Mg2+增加CT的标记，但减少IAP的标记。此外，Mg2+增强鸟嘌呤核苷酸的作用，增加GTP[S]的抑制作用以及GDP[S]或GTP的激活作用。在10 mM MgCl2存在下于30℃预孵育肝膜不可逆地抑制IAP的标记。用4.95 mM N-乙基马来酰亚胺预处理肝膜使CT的标记减少约15%，但几乎完全阻断IAP的标记。这些结果表明，腺苷酸环化酶的抑制性GTP结合蛋白Ni的未解离、结合GDP的构象是IAP进行ADP核糖基化的首选底物。这种构象在天然膜中普遍存在，对温度、Mg2+离子和诸如N-乙基马来酰亚胺的烷基化剂敏感。在30℃时，Mg2+可能导致天然膜中Ni的解离和变性。
