HORMONAL INTERACTION WITH HUMAN ERYTHROCYTE MEMBRANE

Abstract

The polypeptide composition of erythrocyte membrane from haemoglobin AA, AS, SS (Hb-AA, AS, SS) subjects have been quantitatively analysed by sodium dedecyl sulphate electrophoresis. In all membranes, nine main polypeptide bands have been identified on the basis of their apparent muscular weight determination. These polypeptides are, Bands 1 & 2 having a similar gel mobility as spectrin with a molecular weight of 200,000 and 240,000. Other major, polypeptides identified correspond to band 3 (M.W 90,000), band 4.5 (M.W 50-65,000) and band 5 (M.W 45,000) band 7 (M.W 50,000) and band 6 which was unique to Hb-SS membranes with a molecular weight of 11,000. There were significant differences in the polypeptide composition of at least five of the compounds, when HB-AA and HB-AS membranes on the one hand were compared to HB-SS membrane. The test abundant polypeptides (band 3) constituted only 10.8% of the total membrane proteins in Hb-SS membranes when compared with 26.8% in Hb-AA and 26.0% in Hb-AS membranes (AA vs SS p<0.01). These changes in Hb-SS membranes could be degradative changes. Incubation with steroid hormones in vitro, did not induce any observable alteration to the reported polypeptide profiles in all membranes. The (Ca2 +Mg2+) Atpase activity in Hb-SS membrane was significantly less than in Hb-AA and Hb-AS membranes (AA 0.76 ± 0.04: AS 0.73 ± 0.04 and Hb-SS 0.29 ± 0.01. P1 mg-1 prot hr⁻¹; AA/SS p<0.05). Generally the (Ca2+ +Mg2+) AATPase activity of AA membrane was not stimulated by any steroid hormones used in contrast to Hb-SS membranes whose activity was significantly enhanced by nearly all the steroid hormones. The stimulation of Hb-SS membranes shows a potency sequence of oestradiol >progesterons> testosterous > cortisol > prognenolone. The differential behavior of Hb-As membranes under stimulation by different steroids is noteworthy. Steroids with 3-OH substituents on their cyclopentane structure, stimulated (Ca²⁺ Mg²⁺) ATPase activity in Hb-AS membranes whereas those with 3-keto (C=0) substituents on their cyclopentane backbone, did not. Iodothyronines and their analogues did not stimulate (Ca²⁺ Mg²⁺) ATPase activity, in all membranes, Hb-SS erythrocytes which were preincubated with various thyroid hormones and analogues, formed fewer sickled cells (5-10%) when compared with saline controls (45%), on deoxygenation. At the same hormone and analogue concentrations, (5x10⁻⁹M; 1x10⁻¹⁰M; 1x10⁻¹⁴M), subsequent incubation of pre-sickled erythrocytes with hormones did not reverse sickling. In this study only adrenaline enhances the stimulation of AA membrane (Ca²⁺ Mg²⁺) ATPase, as well as the highest adenylate cyclase activity and cyclic nucleotide formation in all membranes, in a concentration dependent manner. The mean basal adenylate cyclase activity of Hb-AA membranes was 31.2 ± 6.5, compared to the mean activity of 75.9 ± 7.1 p. mol. cAMP mg⁻¹ in Hb-SS membranes. (p<0.005). There was no apparent correlation between the increased reticulocyte content of sickle cell blood and its higher adenylate cyclase activity. Also whilst sodium fluoride significantly enhanced the adenylate cyclase activity in Hb-AA membranes: Hb-SS membranes did not show such response to NaF stimulation. T₃ and T₄ stimulated adenylate cyclase activity in Hb-AA, AS and SS membranes, but the interaction of the thyroid hormones analogues was not significant. This suggest that the effect of T₃ and T₄ is related to their hormonal activity.