Hexokinase was purified 680-fold from erythrocytes of man. The preparation was not contaminated by other glycolytic enzymes.Initial reaction rate measurements were performed at pH 7.2, ionic strength 0.15 and 37 "C by use of coupled optical assays. The experimental data were used to construct a kinetic model of the enzyme, the kinetic parameters of which were estimated by means of a computer-fit program.The hexokinase of erythrocytes operates by a rapid-equilibrium random mechanism. The dissociation constant of the E . glucose complex is 0.038 to 0.054 mM, of the F, -MgATP complex 1.0 to 2.1 mM. Uncomplexed Mg2+ up to 4 mM is capable of activating the enzyme velocity twofold (Knrgl+ = 1.0 mM). An inhibition was observed a t higher Mg2+ concentrations. a-D-Glucose 6-phosphate and &-D-glUCoSe 1,6-bisphosphate inhibit the hexokinase competitive to MgATP with dissociation constants of 0.069 mM. The enzyme is also inhibited by 2,3-bisphosphoglycerate competitive with respect to MgATP with a K P~G = 2.7 mM. An inhibition by uncomplexed ATP was not detected. The parameters of the kinetic model of the enzyme in conjunction with the free intracellular Concentrations of substrates and effectors were used to calculate the actual intracellular activity of the hexokinase in dependence on variations of pH, temperature and the state of haemoglobin oxygenation. The calculated hexokinase activities correspond to the glycolytic flux in all conditions considered, except a t pH 8.

Under simulated intracellular conditions (pH 7.2, 37 "C, 130 mM KC1, 20 mM NaCl and haemoglobin concentrations > 1.5 mM) the conditional association constants for the binding of ATP and 2,3-bisphosphoglycerate (P,-glycerate) to deoxygenated and oxygenated haemoglobin (Hb and HbO,, respectively) and the influence of Mg2+ on the binding of phosphocompounds were determined by means of a cation-sensitive electrode and ultrafiltration.The KIass for the binding of ATP to Hb is 2.6 mM-l and 0.36 mM-l for HbO,. P,-glycerate is bound to Hb with K a s s of 5 mM-l and to HbO, with a K',,, of 0.25 mM-l. The anions compete with each other in binding. Mg2+ is not bound to haemoglobin but it reduces the binding of the phosphocompounds by complexing with them. It decreases the shift of the oxygen binding curve produced by ATP. MgATP is weakly bound to Hb with a K a s s of 0.14 mM-l and to HbO, with a K'a,, of 0.039 mM-l. Binding of the Mg P,-glycerate complex to haemoglobin was not established.The calculated values for the dependence of p5,, (the partial pressure of oxygen a t half-satwation of haemoglobin) on the free concentration of P,-glycerate, based on the K a s s of Hb and HbO, complexes, agree well with the experimental data on erythrolysates and intact human red blood cells. The K'ass are furthermore an appropriate basis for the estimation of the intraerythrocatic state of Mg2+ and the metabolites.Haemoglobin binds polyvalent anions including the phosphometabolites in a reversible manner. The free concentrations of magnesium, nucleotidcs and of their complexes are influenced by binding. 2,3-Bisphosphoglycerate (P,-glycerate) which accounts for about half of the total acid-soluble phosphate in human erythrocytes [l], influences the concentration of free MgATP both by competition with ATP in the binding of haemoglobin and by complexing magnesium ions.Two phosphotransferases, e.g. hexokinase and phosphofructokinase, function as the main controlling enzymes in the glycolytic sequences of red blood cells 121. Magnesium -nucleotide complexes are substrates as well as modifiers of these enzymes. AnAbbreviations. P,-glycerate, 2,3-bisphosphoglycerate; HbO, oxygenated haemoglobin; Hb, deoxygenated haemoglobin; K,,,, association constant.Enzymes. Hcxokinase (EC 2.7

The intracellular distribution of ATP, 2,3-bisphosphog1ycerate(P2-g1ycerate) and Mg2+ was calculated for the oxygenated and deoxygenated human erythrocyte for the normal range and that of pathophysiological variations based on the association constants of the relevant complexes.The data indicate that about 2001, of ATP is bound both in the oxygenated and deoxygenated cells, while 39 and 7301, of P,-glycerate is bound under these conditions. An increase of the free Mg2+ concentration from 0.7 to 1.1 mM is produced by complete deoxygenation of haemoglobin.The calculations would indicate that during deoxygenation of haemoglobin the hexokinase reacts to the increased concentration of the activator Mg2+ and the decline of the inhibitor P,-glycerate with an elevation of its activity which corresponds to experimental data on intact erythrocytes.The P,-glycerate formation rate in deoxygenated cells is stimulated about 2.5 times in comparison to oxygenated cells as estimated from the free concentration of P,-glycerate and the kinetic constants of bisphosphoglycerate mutase.An assessment of the possible influence of other anions including bicarbonate shows that the distribution of the species of ATP, P,-glycerate and Mg2+ are changed by less than 20°/,.Together with the data presented in the accompanying paper, the results given here indicate that the constants and estimates are approximately valid for intracellular conditions. I n the preceding paper [I] the interactions among ATP, 2,3-bisphosphoglycerate (P,-glycerate), Mg2+ and oxygenated and deoxygenated hemoglobin (HbO, and Hb, respectively) were reported. Association constants valid for intra-erythrocytic conditions were given.Glycolysis depends on the state of the metabolites within the cell; only their forms not bound to haemoglobin or other proteins are substrates and effectors in the enzymatic reactions. These forms enter in the mass action equations. Both ATP and P,-glycerate 2.2.1.1).play an important role in the regulation of glycolysis. A model of the regulation of the glycolytic chain in erythrocytes therefore requires the knowledge of the binding of these compounds.It is the aim of the present communication to describe quantitatively the intracellular distribution of the various species of ATP, P,-glycerate and Mg2+ for the oxygenated and deoxygenated cell based on the association constants of the relevant complexes. The range of variation for ATP, P,-glycerate and Mg2+ was chosen so as to cover both the normal range and that of pathophysiological variations. The data were compared with an assumed haemoglobin-free cell. CALCULATIONS

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The implication of activated oxygen in the interaction between hypoxic rat liver and circulating erythrocytes was investigated. Reduced species of oxygen generated in hypoxic liver owing to accelerated purine nucleotide degradation via xanthine oxidase initiate alterations of plasma membrane and glutathione system of erythrocytes. Osmotic fragility, hemolysis rate and erythrocytic GSSG:GSH ratio may be considered as appropriate indicators of oxidative load in liver and other tissues. Addition of erythrocytes to the perfusion medium attenuates the GSSG efflux of hypoxic liver from 2.7 +/- 0.5 nmol x g w.w.-1 x min-1 to 1.4 +/- 0.2 nmol x g w.w.-1 x min-1 Thus, circulating erythrocytes protect the liver against oxidative attack.