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Abstract
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 requires magnesium, is accelerated by isoproterenol, and is inhibited by GTP or sodium fluoride. The conditions for preactivation resemble those for the specific binding of Gpp(NH)p to the regulatory component of adenylate cyclase and consequent quasi-permanent activation of the cyclase, except that direct activation of the cyclase does not depend upon a cytosolic macromolecule. We conclude that the substrate for cholera toxin is a complex of the GTP-binding protein with GTP, in some way modified by a macromolecule of the cytosol.
Highlights
From the Department of Biology, Harvard University, Cambridge, Massachusetts02138, and Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts 02111
Cholera toxin-catalyzed ADP ribosylation of the supplied to intact cells the toxin binds toganglioside G Mo~n membrane-bound GTP-binding protein that regulates the cell surface and subsequently releases its active fragment adenylate cyclase activity requires certaincomponents Al into thecytoplasm
The injected fragment AI catalyzes an of the cytosol
Summary
Stood,but in view of itsregulatory rolein theadenylate cyclase system it habseen attractive to think that GTP might act by inducing some conformationalchange that enabled the GTP-binding protein to serve assubstrate for cholera toxin. The suspension was incubated for 30 min at 37"C, and the of the GTP-binding protein(Mr42,000) and for the concomitant activation of adenylate cyclase. Cyclase activities are quoted as picomoles of cyclic AMP. One microliter of packed ghosts contains about pg of membrane protein
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