RESUMEN
The H(+)-ATPase from Schizosaccharomyces pombe belongs to the group of transport ATPases which displays two main conformational states, E1 and E2 (P-type ATPase). In this report, we show that, as in the case of other P-type ATPase, the purified enzyme exhibits a p-nitrophenylphosphatase activity which can be completely inhibited by vanadate. In aqueous medium, p-nitrophenyl phosphate hydrolysis proceeds at only 0.5% of the rate of ATP hydrolysis, and both activities can be stimulated 3- to 4-fold by decreasing the pH from 7.5 to 6.5. Addition of the organic solvent dimethyl sulfoxide (10-40%), which has been shown to favor the E2 conformation, stimulates the p-nitrophenylphosphatase activity but inhibits the ATPase activity. At pH 7.5, the Km for p-nitrophenyl phosphate decreases when dimethyl sulfoxide is present. In the presence of 30% (v/v) dimethyl sulfoxide, the phosphatase activity can be inhibited by ATP (K(i) 300 microM) or by P(i) (K(i) 1 mM). The H(+)-ATPase incorporated into liposomes retains pNPPase activity, but it does not support H+ transport. Gel electrophoresis reveals that the pattern of H(+)-ATPase cleavage by trypsin changes when vanadate, Me2SO, or both compounds are present in the medium, regardless of the pH used during trypsinization. We propose that p-nitrophenyl phosphate is hydrolyzed by a H(+)-ATPase conformation distinct from that which hydrolyzes ATP, most probably an E2-like form. We also suggest that, in addition to the E1-E2 transition, the enzyme activity can be regulated by protons at another step of the catalytic cycle.
Asunto(s)
4-Nitrofenilfosfatasa/metabolismo , Membrana Celular/enzimología , ATPasas de Translocación de Protón/metabolismo , Schizosaccharomyces/enzimología , 4-Nitrofenilfosfatasa/antagonistas & inhibidores , 4-Nitrofenilfosfatasa/aislamiento & purificación , Dimetilsulfóxido/farmacología , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Cinética , Liposomas , Peso Molecular , Fragmentos de Péptidos/aislamiento & purificación , Fosfatos/farmacología , ATPasas de Translocación de Protón/aislamiento & purificación , Tripsina , Vanadatos/farmacologíaRESUMEN
The activation of the Ca(2+)-ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the concentration of effector required to elicit half-maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin-Sepharose resin and in dependence on calmodulin concentrations when the pH is increased. In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin-binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.