Potassium activated phosphatase from human red blood cells. The effects of p-nitrophenylphosphate on carbon fluxes.

1. When red cells are incubated in solutions containing p-nitrophenyl-phosphate (p-NPP), intracellular p-NPP quickly builds up reaching with a half-time of 3 min a concentration in cell water equal to one fourth the external concentration, which under the conditions used is the expected value for a divalent anion in Gibbs-Donnan equilibrium. Hence p-NPP added to the incubation media in red cells has quick access to the active centre of the membrane phosphatase which is located at the inner surface of the cell membrane.2. When p-NPP is added to the incubation media of ATP-free red cells or reconstituted ghosts, no ouabain-sensitive cation movements are detectable, suggesting that hydrolysis of p-NPP by the active transport system is unable to energize active ion translocation.3. When p-NPP concentration in the incubation media of ATP-containing cells is progressively raised, both ouabain-sensitive Na loss and ouabain-sensitive Rb uptake tend to zero along rectangular hyperbolae. For both movements inhibition is half-maximal at 77 mM external p-NPP (i.e. 19 mM internal p-NPP).4. p-NPP inhibits with equal effectiveness the Na:K and the Na:Na exchanges catalysed by the Na pump.5. The inhibitory effect of p-NPP cannot be attributed to the products of its hydrolysis, is inversely related to the intracellular ATP concentration and seems to be exerted at the inner surface of the cell membrane with an apparent affinity similar to that of the membrane phosphatase. These facts suggest that inhibition is mediated by the combination of p-NPP with the active centre of the membrane phosphatase.6. Apart from affecting the ouabain-sensitive cation movements, p-NPP increases the ouabain-resistant uptake and loss of both Na and Rb. This effect is about 4 times larger for Rb than for Na, and its kinetic analysis suggests that it is due to an increase in the passive permeability of the cell membrane.7. The increase in passive cation permeability upon addition of p-NPP cannot be attributed to the products of its hydrolysis. It seems to be due to the combination of p-NPP with a site which, like the active centre of the ouabain-resistant membrane phosphatase, faces the inner surface of the cell membrane, is unaffected by ATP and is half saturated by about 15 mM-p NPP.