Orthophosphate (Pi) uptake was examined in human red blood cells at 37~ in media containing physiological concentrations of Pt (1.0-1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene-2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/P i cotransport pathway determined the KI/~ for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na ([Pi]o = 1.0 mM) to be 304 • 24/zM and 139 • 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a V~ of 0.63 _+ 0.05 mmol Pi (kg Hb)-l(h) -1 at 140 mM Na o. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The V=~, of the Na/P i cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/P i stoichiometry of 1.73 • 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular S~P i labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate. These results demonstrate the existence of a Na/Pi cotransport system in the red cell membrane that is important in Address reprint requests to Dr.

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The interaction of the cardiac glycoside [3H]ouabain with the Na+,K+ pump of resealed human erythrocyte ghosts was investigated . Binding of [3H]ouabain to high intracellular Na' ghosts was studied in high extracellular Na' media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na +,K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na +,K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate . Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A . Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+,K+ pump (K m -1 jLM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-,8-y-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor .