Kinetic alterations of the red cell membrane phosphatase in α‐ and β‐Thalassemia

Abstract

We studied the red cell membrane neutral phosphatase, which is part of the Na+K+ ATPase, in several types of oxidative hemolytic anemias. We used an artificial substrate, the p-nitrophenylphosphate. In controls and in patients heterozygous for various unstable hemoglobins (Hb Hope, Hb Köln, or Hb Hammersmith), the kinetics were of the Michaelis-Menten type. On the contrary, in nearly all patients with alpha- or beta-thalassemia, the kinetics displayed an abnormally biphasic character. The apparent Michaelis constant (KMapp) was significantly decreased. The biphasic character correlated with the imbalance of globin chain synthesis. The beta-mercaptoethanol markedly increased Vmax in controls, but had little effect on the biphasic kinetics. Omission of K+ abolished the biphasic kinetics. The abnormal kinetics failed to appear with another artificial substrate, the 4-methylumbelliferylphosphate, nor did it appear with ATP, the natural substrate. In vitro, H2O2 treatment of normal and thalassemic red cells was unable to induce or exaggerate, respectively, the biphasic kinetics, but generated alterations of a different nature. We suggest that the various kinetic alterations of the phosphatase in thalassemic syndromes originate from the imbalance of globin chain synthesis. However, the involvement of an oxidative process remains to be demonstrated.

Increased tyrosine phosphorylation of band 3 in hemoglobinopathies

“…The effect of hydrogen peroxide on band 3 tyrosine phosphorylation is of interest since in SS cells, the antioxidant defenses are impaired and they spontaneously generate twice the normal amount of activated oxygen species [40]. Membrane-bound pnitrophenylphosphatase activity has also been described and was observed to be depressed or altered in membranes of individuals with sickle cell disease, congenital hemolytic anemias, and thalassemias [41][42][43]. Several intracellular changes in thalassemic erythrocytes could account for the oxidative damage in their membrane components since oxidation of excess alpha-hemoglobin subunits and intracellular excess of iron result in increased formation of free oxygen radicals [44].…”

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“…It is possible that oxidative stress affects β-spectrin phosphorylation by modification of spectrin kinases/phosphatases, or by alterating the spatial arrangement of membrane proteins. [18][19][20][21][22] Reduced tyrosine kinase activity of membrane proteins is observed in Scott syndrome, a hereditary bleeding disorder characterized by a deficiency in prothrombinase activity caused by defective phospholipid scrambling activity of the blood cell membrane. 23 The decrease in membrane phospholipid scrambling results in reduction in phosphatidyl serine (PS) exposure and in prothrombinase activity on the cell surface.…”

The Hb F composition in a Moroccan Family with β°‐thalassaemia and Hb O‐Arab

“…Erythrocyte ghosts were prepared essentially according to Dodge et a1 (16). The kinetics parameters of the membrane neutral phosphatase were determined as previously described, after two additional washes of the ghosts with 5 mM Tris (pH 7.50) (17,18).…”

“…The membrane alterations in the Hb 0-Arab-fP-thalassaemia association (Figure 4) The kinetics of the membrane neutral phosphatase was typically biphasic in 1.2, with a reduced KMapp (1.58 mM") and an increased Vrnax (6.44 nmol mg-' min-'"), the latter being due to the rejuvenation of the red cells (17). This kind of kinetics reproduces that observed in @thalassaemia major, including the variety resulting from the Hb E/@-thalassaemia combination (17).…”

“…The membrane alterations in the Hb 0-Arab-fP-thalassaemia association (Figure 4) The kinetics of the membrane neutral phosphatase was typically biphasic in 1.2, with a reduced KMapp (1.58 mM") and an increased Vrnax (6.44 nmol mg-' min-'"), the latter being due to the rejuvenation of the red cells (17). This kind of kinetics reproduces that observed in @thalassaemia major, including the variety resulting from the Hb E/@-thalassaemia combination (17). The kinetics was normally monophasic in all other cases, however KMapp was reduced not only in 11.1 and 11.3, carrying a fi-thalassaemia trait (0.91 and 1.14, respectively), but also in 11.2 and 11.4, displaying heterozygous Hb 0-Arab (1.26 and 1.51, respectively).…”