A COMPARATIVE STUDY OF THE STRUCTURES OF THE RED CELL MEMBRANES AND THE CALCIUM-PUMPING ADENOSINE TRIPHOSPHATASE IN SOME MAMMALIAN SPECIES

Abstract

Erythrocyte ghost membranes were isolated from blood samples obtained from five mammalian species, namely: (i) human (Homo sapiens), (ii) bovine (Bos taurus), (iii) porcine (Sus scrofa melitensis), (iv) ovine (Ovis aries crassicandus), and (v) caprine (Capra hircus syriaca). The major polypeptide components of these plasma membranes were separated by the sodium dodecyl sulphate polyacrylamide gel electrophoretic technique and stained with Coomasie Brilliant Blue, The relative amounts of the various polypeptides vary from species to species. Porcine erythrocytes have greater amounts of band 2.3 (one of the spectrin-binding proteins) than human, bovine and ovine erythrocytes, while caprine erythrocytes lack this band totally. Band 3 in the human erythrocyte membrane is a diffuse and broad band, but it appears as a narrow band in the other species, suggesting a greater degree of homogeneity in these species. Two unidentified bands, absent in the other species, are seen in the neighbourhood of band 6 in porcine erythrocyte membranes. Bands 7 and 8 are not well separated in ovine and caprine erythrocyte membranes. These could possibly represent a split or diffuse band 7 rather than two distinct bands in these species. The kinetic properties of the membrane-bound calcium-pumping protein, Ca2+, Mg2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were also compared in the five mammalian species. The specific activities of the enzyme range from 0.36 ± 0.02 µmoles/mg. protein/hr. in human erythrocytes to 2.86 ± 0.06 µmoles/mg. protein/hr, in porcine erythrocytes. The membrane bound enzyme has a Vmax of 0.38 ±- 0.06, 0.36 ± 0.06, 6.25 ± 0.53, 1.05 ± 0.28 and 0.42± 0.11 µmoles/mg. protein/hr. in human, bovine, porcine, ovine and caprine erythrocytes respectively. Corresponding apparent Km(ATP) values are: 0.25 ± 0.12, 0.45 ± 0.18, 0.45 ± 0.11, 0.36 ± 0.12 and 0.31 ± 0.09 mM. The enzyme is highly sensitive to vanadate, K1 values for the vanadate inhibition of the enzyme in the various species are 6.58 ± 0.42, 11.84 ± 0.53, 2.08 ± 0.11, 5.63 ± 0.21 and 8.61 ± 0.38 µM respectively. The effects of 3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-butyric acid (DBA), an antisickling agent, on the Ca2+ , Mg2+ATPase were also studied in the human erythrocyte membrane (normal and sickle cell). DBA activates the enzyme in a concentration-dependent fashion. The concentration-effect studies were carried out between 0.2 and 4.0 mM DBA. A maximal stimulation of 135% is obtained with normal erythrocyte ATPase at 0.8 mM DBA, compared with a maximal stimulation of 81.1% obtained with the sickle cell enzyme at 0.5 mM DBA. These findings indicate that: (i) the Ca2 ± pumping ATPase could be used as a marker for the pig erythrocyte plasma membranes among its ruminant counterparts, and (ii) DBA is a potential drug for the treatment of sickle cell disease.