Characterization of K(+)-dependent and K(+)-independent p-nitrophenylphosphatase activity of synaptosomes.
These experiments examined effects of several ligands on the K+ p-nitrophenylphosphatase activity of the (Na+,K+)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K(+)-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K+ was approximately 75% less than that observed when K+ was included in the incubation medium. The response to the H+ concentrations was different: K(+)-independent activity showed a pH optimum around 6.5-7.0, while the K(+)-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K(+)-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K(+)-independent activity. Na+ did not affect K(+)-independent activity at all, while the same ligand concentration inhibited sharply the K(+)-dependent activity; this inhibition was not competitive with the substrate, p-nitrophenyl phosphate. K(+)-dependent activity was stimulated by Mg2+ with low affinity (millimolar range), and 3 mM Mg2+ produced a slight stimulation of the activity in absence of K+, which could be interpreted as Mg2+ occupying the K+ sites. Ca2+ had no appreciable effect on the activity in the absence of K+. However, in the presence of K+ a sharp inhibition was found with all Ca2+ concentrations studied. ATP (0.5 mM) did not affect the K(+)-independent activity, but this nucleotide behaved as a competitive inhibitor to p-nitrophenylphosphate. Pi inhibited activity in the presence of K+, competitively to the substrate, so it could be considered as the second product of the reaction sequence.
Dpto. de Bioquímica y B. Molecular, Facultad Biología, Universidad La Laguna, Canary Islands, Spain.
M
M
Guerra Marichal
A
A
Rodríguez del Castillo
P
P
Martín Vasallo
E
E
Battaner Arias
D009597:4-Nitrophenylphosphatase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002540:Cerebral Cortex / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013574:Synaptosomes / Q000201:enzymology*
D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium; D002118:Calcium