The interaction of haemoglobin, magnesium, organic phosphates and band 3 protein in nucleated and anucleated erythrocytes

Abstract

The anion influx was measured in order to study the interaction among organic phosphates, magnesium, haemoglobin and the N-terminal of the cytoplasmic domain of band 3 protein in human, chicken and trout erythrocytes. The rate constant for SO(4)(2-) influx in human and trout erythrocytes increased significantly when it was measured with an increased concentration of intracellular Mg(2+). The SO(4)(2-) influx was also measured in human erythrocyte ghosts in the presence and absence of Mg(2+). The smaller activation provoked by Mg(2+) in ghosts could be caused by the presence of a small quantity of haemoglobin which remained inside. The SO(4)(2-) uptake in chicken erythrocytes in the presence and in absence of Mg(2+) was characterized by very similar rate constants. The results suggest that the small increase in intracellular Mg(2+) in the erythrocytes involves an increase in the formation of Mg(2+)-ATP and Mg(2+)-2,3 BPG complexes reducing the affinity of the organic phosphates for Hb. This new situation may influence the functions of the anion transporter with consequent variations of SO(4)(2-) influx throughout the erythrocyte membrane in human and in trout erythrocytes, whereas in chicken RBCs this function cannot occur and, in fact, no increase in sulphate influx was noticeable. The measurement of Hb/O(2) affinity by the use of alternating fixed and variable concentrations of organic phosphates and Mg(2+), confirms the interactions between these elements and their effect on the mechanism of the affinity. When we measured the sulphate influx in the presence of DIDS we found some differences in the three types of cells.

Effects of Nickel on Human and Fish Red Blood Cells

“…At specified intervals, 5 ml samples of the suspension were removed and added to a test tube containing 5 lM 4,4¢-diisothiocyanato-stilbene-2,2¢-disulfonate (DIDS) stopping medium and kept in ice. DIDS, a compound that binds irreversibly and specifically to the band 3 protein, inhibits SO 4 2-transport in both human and fish red blood cells (Lepke et al 1976;Romano et al 2002;De Luca et al 2004). After withdrawal of the last sample, the erythrocytes were washed three times in a sulphate-free medium at 0°C to remove extracellular sulphate, then lysed with 1 ml distilled water and 100 ll trichloroacetic acid at 99%.…”

Oxidative stress and band 3 protein function in Liza aurata and Salmo irideus erythrocytes: effect of different aquatic conditions

“…26 The changes in studied parameters could reflect loss of homeostasis or demonstrate a compensatory response to salinity changes. The aquatic environment plays a relevant role in the control of structural and functional erythrocyte properties, which are the dominant cell type in the blood of most of fish species.…”

“…[24][25][26] After withdrawal of the last sample, the erythrocytes were washed and centrifuged three times in a sulphatefree medium at 0 C to remove extracellular sulphate. At regular intervals, 5-ml samples of suspension were removed and added to a test tube containing 6 ml of 4,40-diisothiocyanostilbene-2,20-disulfonic acid stopping medium and kept in ice.…”

Effects of lead chloride on human erythrocyte membranes and on kinetic anion sulphate and glutathione concentrations

“…where Co, C t and C ∞ represent the intracellular sulphate concentrations measured at times 0, t and , e indicates the Euler's number (2.7182818), and r is a constant [14,15].…”