Abstract
Scopadulcic acid B (SA-B), a novel diterpenoid, is a main ingredient of the Paraguayan traditional medicinal herb "Typychá kuratú (Scoparia dulcis L.). SA-B and its debenzoyl derivative, diacetyl scopadol (DAS), specifically inhibit ATP hydrolysis of gastric H+,K(+)-ATPase. Both compounds inhibit the K(+)-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step. SA-B is a mixed-type inhibitor with respect to the activating cation, K+. SA-B lowers the affinity of H+,K(+)-ATPase to K+ and decreases the maximal velocity of ATP hydrolysis, whereas DAS is an uncompetitive inhibitor with respect to K+. Furthermore, the effects of SA-B and DAS on conformational states of the ATPase were studied by measuring the changes in the fluorescence intensity of the fluorescein isothiocyanate-labeled enzyme. The fluorescence study shows that SA-B primarily binds to the E2K form in the presence of Mg2+ and stabilizes the form and that DAS stabilizes the E2PK form. Therefore, the chemical modification of SA-B, debenzoylation, induced the changes in the pattern of inhibition of H+,K(+)-ATPase. Furthermore, the inhibition mechanisms of SA-B and DAS were different from those of omeprazole, which is an irreversible inhibitor, and SCH 28080, which is a reversible, competitive inhibitor with respect to K+. DAS also inhibited the K(+)-dependent p-nitrophenyl phosphatase activity, and the inhibition was competitive with respect to K+, indicating that the K(+)-dependent p-nitrophenylphosphatase activity does not represent the partial reaction step of H+,K(+)-ATPase.
Highlights
Scopadulcic acid B (SA-B), a novel diterpenoid, is a main ingredient of the Paraguayan traditional medicinal herb “Typychi kuratii” (Scoparia dulcis L.)
We show that scopadulcic acid B (SA-B),’
diacetyl scopadol (DAS) even at 50 p.M inhibited the activity by 40% (Fig. 2B), and we could not measure the effect of DAS at 100,uM, because DAS at concentrations higher than 50 p.M was insoluble in the assay medium
Summary
Materials-SA-B was isolated from Scoparia dulcis L. as described elsewhere [19]. It is a diterpenoid with a novel skeleton as shown in. K+-dependent p-nitrophenylphosphatase (K+-pNPPase) activity was measured in 1 ml of reaction mixture containing gastric vesicles (10 p.g of protein), 6 mM MgSO., 6 mM p-nitrophenyl phosphate, 40 mM Tris-HCI (pH 7.4) in the presence or absence of 15 mM KCI. Fifty micrograms of lyophilized gastric vesicles were incubated in a solution containing inhibitor (SA·B or DAS) at the indicated concentration, 2 mM MgSO., and 40 mM Tris-HCI (pH 7.4) at 25 °C for 20 min. To study the effect of SA-B or DAS on the dephosphorylation step, H+,K+-ATPase in lyophilized gastric vesicles was phosphorylated in the presence or absence of SA-B or DAS as described above, and the mixture was incubated with various concentrations of KCI at 25 °C for 10 s. The maximal volume of ligands added was 0.5% of the total volume in order to avoid dilution effects on the fluorescence intensity
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