Cholera toxin activation of adenylate cyclase. Roles of nucleoside triphosphates and a macromolecular factor in the ADP ribosylation of the GTP-dependent regulatory component.

作者：Keiichi Enomoto, Duncan Michael Gill · 发表于：Journal of Biological Chemistry · 年份：1980 · DOI：10.1016/s0021-9258(19)86022-2 · 被引用次数：147 · 研究领域：Cellular transport and secretion、Endoplasmic Reticulum Stress and Disease、Bacterial Genetics and Biotechnology

Cholera toxin-catalyzed ADP ribosylation of the membrane-bound GTP-binding protein that regulates adenylate cyclase activity requires certain components of the cytosol. In broken cells it is prevented by depletion of endogenous nucleotides and is restored by the provision of GTP (1 to 3 PM half-maximum) or ITP (30 PM) although not by ATP, CTP, TTP, or UTP. An hydrolysis-resistant GTP analog, guanyl-5’-yl imidodiphosphate (Gpp(NH)p, 1 to 3 p ~ ) , also supports the toxin-catalyzed reaction, but adenyl-5’-yl imidodiphosphate is only weakly effective. The ability of nucleoside triphosphates to support toxin-catalyzed ADP ribosylation or the toxin-catalyzed activation of adenylate cyclase correlate well. Even with excess GTP or Gpp(NH)p, the rate of ADP ribosylation and adenylate cyclase activation depends upon the presence of a macromolecular component of the erythrocyte cytosol. If GTP is employed, ADP ribosylation requires the simultaneous presence of this nucleotide, cytosolic macromolecules, NAD’, toxin, and membranes. However, membranes may be predisposed to respond to the toxin by preincubating them with Gpp(NH)p and cytosolic macromolecules. Preincubated membranes, washed free of unbound nucleotide, can then be ADP ribosylated by incubation with cholera toxin and NAD+ without additional cofactors. Both Gpp(NH)p and the macromolecular fraction of the cytosol are required for such preactivation. It is complete in 15 min at 37°C and is about three times slower at 25°C. It re...