p-nitrophenylphosphatase activity of plasma membrane H(+)-ATPase from yeast. Implications for the regulation of the catalytic cycle by H+.
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.
Ferreira-Pereira
A
A
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro-Cidade Universitária, Brazil.
Alves-Ferreira
M
M
de Carvalho-Alves
P C
PC
D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008081:Liposomes; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010710:Phosphates / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology*; D014357:Trypsin; D014638:Vanadates / Q000494:pharmacology
D008081:Liposomes; D010446:Peptide Fragments; D010710:Phosphates; D014638:Vanadates; D009597:4-Nitrophenylphosphatase; D014357:Trypsin; D006180:Proton-Translocating ATPases; D004121:Dimethyl Sulfoxide