The uptake and hydrolysis of p-nitrophenyl phosphate by red cells in relation to ATP hydrolysis by the sodium pump.

Abstract

1. The hydrolysis of p-nitrophenyl phosphate has been studied in human red cells, ghosts and haemoglobin-free membranes to see whether hydrolysis was related to the functioning of the sodium pump.2. The cell membrane restricted p-nitrophenyl phosphate entry into cells and was rate-limiting for hydrolysis by the large amount of intracellular soluble phosphatase. The uptake was inversely proportional to the external chloride concentration, and inhibitors (phloretin and persantin) of inorganic phosphate uptake also reduced p-nitrophenyl phosphate uptake and hydrolysis. The entry mechanism of p-nitrophenyl phosphate appears to be similar to that of inorganic phosphate.3. p-Nitrophenyl phosphate was hydrolysed in cells almost entirely by ouabain-insensitive phosphatases, both soluble and membrane bound. In ghosts containing less soluble enzyme than cells there was still no component of p-nitrophenyl phosphate hydrolysis that was related to the sodium pump in being sensitive to external potassium or ouabain.4. Haemoglobin-free membranes lacking soluble p-nitrophenyl phosphatase required potassium for optimum p-nitrophenyl phosphate hydrolysis and this part was inhibited by ouabain as was also ATP hydrolysis by the sodium pump. The rates of potassium-dependent and potassium-independent hydrolysis were each increased about threefold on decreasing the electrolyte concentration from 150 to 25 mM. The response was found whether the main electrolyte was potassium chloride, sodium chloride, choline chloride or Tris chloride. In contrast, the changes were not found when the osmotic pressure was varied to the same extent with non-electrolytes. p-Nitrophenyl phosphate hydrolysis was thus activated on lowering the ionic strength. The soluble enzyme was similarly affected and the effect was reversible.5. The membrane ATPase activity was unaffected by the changes in ionic strength which markedly altered p-nitrophenyl phosphate hydrolysis.6. These results with red cell membranes show that the ionic strength under physiological conditions largely prevents the hydrolysis of p-nitrophenyl phosphate, but not of ATP. A possible mechanism is discussed in terms of the effect of different structures of water in electrolyte and non-electrolyte solutions on the enzymic activity of the sodium pump.