Abstract
The functional consequences of Arg-242 to Ser or Lys substitutions in type I alpha regulatory (R) subunits of cAMP-dependent protein kinase were analyzed by using recombinant murine R subunits expressed in Escherichia coli. These mutations arose in cAMP-resistant mutants to S49 mouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding sites. Binding of cAMP to site A of the mutant R subunits could be detected by cAMP-dependent quenching of endogenous tryptophan fluorescence, [3H]cAMP binding to mutant R subunits with the Arg-242 mutations without or with an inactivating mutation in site B, or biphasic dissociation of [3H]cAMP from the mutant subunits at low temperature. The mutations reduced site A affinities by about 25-fold, and the reductions were attributable to accelerated rates of cAMP dissociation. While the presence of cAMP in site A retards dissociation of [3H]cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of [3H]cAMP from site B of the mutant subunits. The predominant effect of the mutations, therefore, was loss of allosteric coupling between the two cAMP-binding sites. A second allosteric interaction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutations at Arg-242.
Highlights
The functional consequencesof Arg-242to Ser or Lys tabolite activator protein(CAP),t’he CAMP-binding protein of substitutionsintype Ia regulatory (R) subunits of Escherichia coli
It be detected by CAMP-dependent quenching of endogenous tryptophan fluorescence[,‘HICAMPbindingto mutant R subunits with theArg-242 mutations withoutor with an inactivating mutationsinte B, or biphasic disremainsunknown how interactions of CAMP withthe conserved arginines and/or other binding site residues result in such dramatic decreases in theaffinity of R for C subunit
Functional manifestations of the allosteric interactions among R subunit, CAMP,and C subunit include the following: decreases in bothon and off rates for CAMP-bindingto siteB mediated by Allosteric Coupling of Cyclic AMP-biSnidteisng
Summary
Subunit preparation) were purified by washing 2500 x g pellet fractions (above)by two to three cycles of resuspension and recentrifugation in Materials. Purification of R Subunits-R subunits were extracted from frozen tation with ammonium sulfate as described elsewhere (Dbskeland and pellets of induced bacteria (from 1to 2 liters of cultures) by thawing, Ogreid, 1988).For the equilibrium binding studies of Fig. 2, about 1.6 suspending with 15 mU1,OOO OD,B, units of EB, and passing twice pmolof R subunit CAMP-binding sites were incubated with various through a French pressure cell at 16,000 lblin'; the extracts were concentrations of [3H]cAMPin 0.1-3.0-ml dissociation buffer for[2] h at centrifugedat 4 "C for 20min at about 2,500 xg. The precipitated proteins were collected by cen- buffer or high salt dissociation buffer containing 1-10 mM unlabeled trifugation, redissolved ina small volume of EB containing 30 mM CAMP,and sampled at various times for bound radioactivity.For sodium chloride, and dialyzed overnight against about 80 volumes of the rapid exchange oft3H1cAMPfrom site A of mutant R subunits, this same buffer. “Diff.”values were determined from the difference spectra minus and plus CAMP
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
