AU-1421

Membrane-bound Na+,K(+)-ATPase (0.1 mg/ml) was incubated with the K(+)-site-directed probe (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421) (Takada, J. et al. (1990) Biochim. Biophys. Acta 1037, 373-379) at 37 degrees C for 30 min in the absence of ligands, then the Na(+)-dependent phosphorylation level was examined in the presence of 10 microM [32P]ATP at 0 degrees C. The level was decreased to 50% and 0% by about 50 microM and 100 microM AU-1421, respectively. Addition of 1 mM K+ during the treatment with AU-1421 resulted in complete maintenance of the phosphorylation level. When the preincubation was performed at 0 degrees C for 10 s, even 100 microM AU-1421 did not impair the phosphorylation. In contrast to the non-phospho form of the enzyme, the K(+)-sensitive phosphoenzyme formed from ATP was immediately inhibited by the addition of AU-1421 at 0 degrees C. The reactivity of the inhibited phosphoenzyme was restored by the addition of K+. About 1 mM K+ gave the same maximal reactivity in the presence of various fixed concentrations (8-41 microM) of AU-1421, but the apparent affinity for K+ decreased simply with the increase of AU-1421 concentration. From this simple competitive relationship, the apparent Ki value of AU-1421 for the phosphoenzyme was calculated to be 7.2 microM. Compared to the non-phospho form of the enzyme, the phospho form appears to be rather susceptible to AU-1421, probably because the K(+)-site of the phosphoenzyme is exposed to the extracellular aqueous phase.

A novel pyridine derivative, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidonopyridine hydrochloride, AU-1421, was found to produce reversible inhibition of the dog kidney sodium and potassium ion-dependent adenosine triphosphatase [(Na,K)-ATPase] with I50 values of about 50 microM. The reversible inhibition was observed when the enzyme was added directly to the enzyme assay media in the presence of saturating concentrations of the enzyme ligands, Na+, K+, Mg2+ and ATP ("turnover conditions"). In the present study, we focused on the reversible inhibition without preincubation of the enzyme with AU-1421. This inhibition was competitive with respect to K+. The K(+)-pNPPase activity of the same preparation was also inhibited by AU-1421 with I50 values of about 90 microM, and this manner was also competitive with respect to K+. ATP enhanced the AU-1421 inhibition of (Na,K)-ATPase, suggesting that AU-1421 also bound to the enzyme-substrate complex. AU-1421 inhibition of (Na,K)-ATPase was not antagonized by ouabain, suggesting the difference of the binding sites between AU-1421 and ouabain. It is therefore proposed that AU-1421 reversibly interacts at or near the K+ site during turnover conditions.