RESUMEN
Gastric H+,K+-ATPase consists of alpha- and beta-subunits. The catalytic alpha-subunit contains a very unique structure consisting of lysine and glycine clusters, KKK(or KKKK)AG(G/R)GGGK-(K/R)K, in the amino-terminal cytoplasmic region. This structure is well conserved in all gastric H+,K+-ATPases from different animal species, and was postulated to be the site controlling the access of cations (or proton) to its binding site. In this report, we studied the role of this unique structure by expressing several H+,K+-ATPase mutants of the alpha-subunit together with the wild-type beta-subunit in HEK-293 cells. Even after replacing all the positively-charged amino acid residues (six lysines and one arginine) in the cluster with alanine or removing all the glycine residues in the cluster, the mutants preserved the H+,K+-ATPase activity, and showed similar affinity for ATP and K+ as well as similar pH profiles as those of wild-type H+,K+-ATPase, indicating that the cluster is not indispensable for H+,K+-ATPase activity and not directly involved in determination of the affinity for cation (proton).
Asunto(s)
Mucosa Gástrica/enzimología , Glicina/química , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Lisina/química , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Cationes/farmacocinética , Línea Celular , Membrana Celular/enzimología , ADN Complementario , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Regulación Enzimológica de la Expresión Génica , Glicina/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , Humanos , Concentración de Iones de Hidrógeno , Riñón/citología , Lisina/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/fisiología , Potasio/farmacocinética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Diterpenos/farmacología , Mucosa Gástrica/enzimología , Animales , Proteínas de Transporte de Catión , ATPasa Intercambiadora de Hidrógeno-Potásio , Riñón/enzimología , Cinética , Medicina Tradicional , Modelos Teóricos , Estructura Molecular , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Porcinos , Valinomicina/farmacologíaRESUMEN
The structure of scopadulcic acid B (2, SDB), a major ingredient of the Paraguayan herb "Typychá kuratu" (Scoparia dulcis L.), was elucidated mainly by comparison of its spectral data with that of scopadulcic acid A (1). SDB inhibited both the K(+)-dependent adenosine triphosphatase (ATPase) activity of a hog gastric proton pump (H+, K(+)-ATPase) with a value of 20-30 microM for IC50 and proton transport into gastric vesicles. Pharmacokinetic studies of SDB in rats indicated that plasma SDB concentrations after i.v. injection of the sodium salt of SDB (SDB-Na) were described reasonably well by a two-compartment open model with Michaelis-Menten elimination kinetics. Plasma concentrations after oral administration of SDB-Na or SDB showed a much slower decline than what was expected following the i. v. study. It was suggested that the sustained plasma level of SDB after oral administration of SDB-Na or SDB was accounted for by relatively slow but efficient gastro-intestinal absorption in rats.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antivirales/química , Diterpenos/química , Plantas Medicinales/análisis , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Diterpenos/farmacocinética , Diterpenos/farmacología , ATPasa Intercambiadora de Hidrógeno-Potásio , Masculino , Paraguay , Ratas , Ratas Endogámicas , Estómago/efectos de los fármacos , Estómago/enzimología , Porcinos , Difracción de Rayos XRESUMEN
Allylisothiocyanate (ANCS) is shown to inhibit (H+ + K+)-ATPase isolated from the parietal cell of hog gastric mucosa. The ATPase is the proton pump in the secretory membrane of the parietal cell and is an essential component of the acid secretory mechanism. Furthermore, ANCS suppresses acid secretion by bullfrog gastric mucosa in vitro. We suggest that one aspect of the stomachic function produced by ANCS-including foods is the suppressive effect of ANCS on the acid secretory mechanism.