Abstract
We have screened various alkyl- and arylguanidinium derivatives as possible competitors of Na+ or Rb+ for the cation sites on renal Na+,K(+)-ATPase. Alkyl-monoguanidinium or alkylbisguanidinium (BisG) compounds (chain lengths of C3 to C10) competitively inhibit the occlusion of Rb+ and Na+ with an order of affinities C10 greater than C8 greater than C6 greater than C4 greater than C3. BisG compounds are approximately twice as effective as the equivalent alkylmonoguanidinium compounds. In media of high ionic strength, affinities of tens of micromolar are observed, e.g. 26 microM for BisG 8. m-(mXBG)- and p-xylylenebisguanidinium were synthesized and were found to compete with Rb+ or Na+ with intrinsic affinities of 7.7 and 8.2 microM, respectively. The hydrophobicity rather than the degree of proximity of the guanidinium groups in all BisG compounds appears to determine the binding affinity. A systematic search has been made of conditions in occlusion assays for which the inhibitor affinities are highest. When the pH is raised from 7.0 to 8.5, a 5-fold increase in affinity is observed, suggesting that the guanidinium derivatives compete with protons at sites of pKa approximately 7.5. Replacing Tris-HCl with choline chloride-containing media raised apparent affinities approximately 2-fold. All guanidinium derivatives stabilize the E1 conformation of fluorescein-labeled Na+,K(+)-ATPase, acting as competitive Na+ analogues. In media containing only 1 mM Tris-HCl, pH 8.55, very high affinities were observed for binding to the fluorescein-labeled enzyme (e.g. 0.08 microM for mXBG). In very low ionic strength medium, the inhibition was still competitive with Rb+ ions. However, there was also evidence for nonspecific adsorption to the membranes. The following findings show that mXBG, a typical guanidinium derivative, behaves as a Na(+)-like antagonist. (a) It inhibits Na+,K(+)-ATPase activity, competing strongly with Na+ but only weakly with K+ ions. (b) It inhibits phosphorylation from ATP, competing with Na+ ions. (c) Like Na+ ions, it blocks phosphorylation from inorganic phosphate. Based on these results, we propose that the guanidinium group binds to a relatively wide vestibule at the cytoplasmic surface; but, unlike Na+ or K+ ions, it cannot pass into a narrower region of the cation transport path within the membrane. Therefore, it blocks the occlusion and active transport of cations. In the future, high affinity guanidinium derivatives may serve the purpose of locating cation-binding domains of the pump protein after being converted to reactive affinity or photoaffinity covalent labels.
Highlights
We have screened various alkyl- and arylguanidimains of the pump protein after being converted to nium derivatives as possible competitors of Na+or Rb+ reactive affinity or photoaffinitcyovalent labels
The hydrophobicity rather alogues combined with selective proteolytic digestion, sitethan the degroefeproximity of the guanidinium groupsdirected mutagenesis, observations on charge transfer propin allBisG compounds appears to determine thbeinding affinity
When the pH is raised from 7.0 to 8.5, a &fold increase in affinity is observed, sugerties or voltage sensitivity of pumps, and the use of cation analogues as probes in fluorescence or NMR studies are providing some insight for the different cation pumps(see Glynn and Karlish (1990) for a recent review)
Summary
The inhibitory potency of a fixed concentration of BisG 8 (0.2 mM) was tested onthe occlusion at asubsaturating concentration of Rb+ (0.27mM) in the choline chloride medium over a pH range of7.0-9.15 (Fig. 3). Thesepossibilities were medium at pH 8.5, the intrinsic affinities of the meta- and distinguished by looking at the conformational state of the para-derivatives were similar (7.7 and 8.2 PM, respectively) protein using FITC-labeled Na+,K+-ATPase(see Rephaeli et (Table 11)and -3-fold higher as compared with BisG 8, the al. Addition of 0.75 p~ mXBG in the absence of Rb+ resulted in a strong fluorescence enhancement, reversed by 0.5 mM Rb+ (Fig. 4 0 ) ,and Na' ions were able to reverse the fluorescence change (data not shown) These observations show that theprotein is largely in the EPstate in the low ionic strength medium (see Skou and Esmann (1980)). MXBG and Rb' concentration (Fig.5B)confirms competition between mXBG and Rb' under these conditions
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