Phosphate inhibition of the human red cell sodium pump: simultaneous binding of adenosine triphosphate and phosphate.
1. The Na+-K+ exchange carried out by the Na+ pump of human red cell ghosts and the Na+ + K+-dependent adenosine triphosphatase (Na+,K+-ATPase) activity of human red cell membranes are inhibited by MgPO4 rather than by free phosphate; similarly, the substrate for the K+-K+ exchange carried out by the pump is MgPO4 rather than free phosphate. 2. Inhibition of the Na+, K+-ATPase activity by MgPO4 is only partially competitive (mixed type) with ATP, and MgPO4 inhibition of the Na+-K+ exchange measured in Na+-free solutions and in K+-free ghosts which contain ATP at relatively high concentration is partially uncompetitive (mixed type) with external K+. 3. When measurements were made in K+-free ghosts and Na+-free solutions, or when Na+,K+-ATPase activity was measured at high ATP concentrations, inhibition by MgPO4 was non-competitive with cell Na+. This observation is not consistent with the Albers-Post reaction mechanism of the Na+ pump, and suggests the presence of an alternative reaction pathway in which ATP combines with the enzyme before phosphate is released. 4. MgPO4 monotonically inhibited the uncoupled Na+ efflux which occurs in solutions free of both Na+ and K+. The uncoupled efflux seemed to be more sensitive to MgPO4 inhibition than the Na+-K+ exchange. 5. Trinitrophenyladenosine-5'-tetraphosphate stimulated the K+-K+ exchange in the presence of MgPO4, and the characteristics of stimulation by TNP adenosine tetraphosphate were little different from the characteristics of stimulation by trinitrophenyladenosine-5'-triphosphate or -5'-diphosphate. The nucleotide binding site at which K+-K+ exchange is stimulated must be able to accommodate a nucleotide with a linear array of four phosphate groups.
Sachs
J R
JR
Department of Medicine, State University of New York, Stony Brook 11794.
D000255:Adenosine Triphosphate / Q000097:blood*; D001693:Biological Transport, Active / Q000187:drug effects; D003864:Depression, Chemical; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D017616:Magnesium Compounds; D008433:Mathematics; D008954:Models, Biological; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000097:blood / Q000494:pharmacology*; D012964:Sodium / Q000097:blood*; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood
D017616:Magnesium Compounds; D010710:Phosphates; C030781:magnesium phosphate; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium