Unsealed Erythrocyte Membranes Research Articles

Effects of lead on the human erythrocyte membrane and molecular models

Lead has no biological function; however, low, and particularly, high levels of exposure have a number of negative consequences for human health. Despite the number of reports about lead toxicity, very little information has been obtained regarding its effects on cell membranes. For this reason, the structural effects of lead on the human erythrocyte membranes were investigated. This aim was attained by making lead ions interact with intact erythrocytes, isolated unsealed erythrocyte membranes (IUM) and molecular models. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane. The results, obtained by electron microscopy, fluorescence spectroscopy and X-ray diffraction, indicated that (a) lead particles adhered to the external and internal surfaces of the human erythrocyte membrane; (b) lead ions disturbed the lamellar organization of IUM and DMPC large unilamellar vesicles (LUV) and (c) induced considerable molecular disorder in both lipid multilayers, the effects being much more pronounced in DMPC.

Erythrocyte membrane microviscosity and phospholipid composition in lead workers.

The microviscosity and fluidity of erythrocyte ghost membranes from lead workers and control subjects was measured by fluorescence polarisation using the fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). Increased lead was associated with a significant decrease in the average microviscosity of resealed and unsealed erythrocyte membranes. Since DPH fluorescence reflects the organisation of lipids in the central core of the membrane, two aspects of phospholipid metabolism were investigated. Phospholipids were extracted from red blood cell ghost membranes and identified by high performance liquid chromatography. The ratio of phosphatidyl choline to phosphatidyl ethanolamine, an established correlate of membrane fluidity, was significantly increased in lead workers. This is attributed to the known increases in red blood cell cholesterol in lead workers and the structural incompatibility of phosphatidyl ethanolamine and cholesterol, which result in a compensatory increase of phosphatidyl choline. Erythrocyte ghost membranes from control subjects were resealed with the intermediates in phospholipid synthesis that increase with a lead inhibited decrease in red blood cell pyrimidine 5'-nucleotidase. Membrane fluidity was not modified by incubation with cytidine triphosphate, uridine triphosphate, cytidine diphosphate choline, or cytidine diphosphate ethanolamine. Alterations in the microviscosity of the lipid regions of the hydrophobic core of the erythrocyte membrane bilayer and in the phospholipid composition of the membrane may be defects which contribute to the clinical and biochemical instability of the red blood cell on exposure to lead.

Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane.

Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.

Impairment of the calcium pump of human erythrocytes by divicine

Divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, produces a remarkable and consistent inactivation of the Ca 2+-ATPase activity of the erythrocyte calcium pump. The patterns of inactivation are similar in normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. Inactivation of Ca 2+-ATPase is apparently unrelated to the cellular GSH system, to the proteolytic machinery of mature erythrocytes, and to calmodulin, and also occurs in hemoglobin-free, unsealed erythrocyte membranes at 50–100 μ m concentrations of divicine. Analysis of erythrocytes that have escaped destruction during the acute hemolytic crisis of a number of favic patients revealed a dramatic elevation of erythrocyte calcium and a significant decrease of Ca 2+-ATPase activity. These results support the view that divicine plays a toxic role in the pathogenesis of favism and suggest that acute electrolyte imbalances, mostly affecting calcium homeostasis, are involved in the mechanisms of erythrocyte damage and destruction in this hemolytic disease.

Regulation of the shape of unsealed erythrocyte membranes By Mg-ATP and Ca 2+

In the presence of MgCl 2 and ATP, the specific viscosity of suspensions of unsealed freezethawed erythrocyte membranes decreased slowly with time at 37 °C. The decrease in viscosity was found to be an index of Mg-ATP-specific induced folding of these membranes. Mg-ATP-dependent shape or viscosity changes were found to be highly temperature dependent and the viscosity of these membranes did not decrease in the presence of 2 m m 5′-adenyl imidodiphosphate and MgCl 2. Cyclic AMP, NaCl, or KCl did not have any effect on the rate of Mg-ATP-induced viscosity decreases. The Mg-ATP-dependent viscosity decreases were inhibited 100% by 1 m m chlorpromazine or 1 m m N-ethylmaleimide. Mg-ATP-dependent viscosity decreases were half-maximally inhibited by 1 μ m Ca 2+ and completely inhibited by 3–5 μ m Ca 2+. Ca 2+ (5 μ m) also inhibited Mg 2+-dependent phosphorylation 25 to 30% in these membranes. However, if these membranes were preincubated in the absence of Ca 2+ for greater than 10 min at 37 °C, 5 μ m Ca 2+ no longer inhibited Mg-ATP-dependent viscosity decreases and only inhibited Mg 2+-dependent phosphorylation 5% in these preincubated membranes. Preincubation of these membranes at 37 °C for 10 min in the absence of Ca 2+ also resulted in the loss of approximately 40 to 50% of the high-Ca 2+ affinity Ca + Mg-ATPase activity. The presence of 5 μ m Ca 2+ in the preincubation medium protected against the loss of the inhibitory effect of Ca 2+ on Mg 2+-dependent phosphorylation and Mg-ATP-dependent viscosity decreases. The presence of Ca 2+ in the preincubation medium also protected against the loss of Ca + Mg-ATPase activity in these membranes. It is hypothesized that freeze-thawed erythrocyte membranes contain a Ca 2+ phosphatase activity which is temperature labile in the absence of Ca 2+ and that this Ca 2+ phosphatase activity may be involved in the regulation of shape of these membranes. Also discussed is the possible relationship of this Ca 2+ phosphatase with Ca + Mg-ATPase activity and the problems inherent in studying Ca 2+-regulated functions in freeze-thawed erythrocyte membranes.