Calcium binding by human erythrocyte membranes

1. The characteristics of Ca²⁺ binding to haemoglobin-free human erythrocyte membranes were investigated by using ⁴⁵Ca and centrifugation partition of `ghosts' from their external incubation medium. Equilibrium of `ghosts' with external Ca²⁺ required less than 15min. 2. The binding did not vary with temperature in the range 0–37°C. 3. At pH7.4 `ghosts' bound a maximum of 283μmol of Ca<sup>2+</sup>/g of `ghost' protein, equivalent to 6.85×10⁷ Ca²⁺ ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca²⁺ binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca²⁺ bound. 6. Extraction of ⁴⁵Ca-labelled `ghosts' with chloroform–methanol showed that the distribution of Ca²⁺ was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca²⁺ (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca²⁺: phosphorus ratio of about 1:2.     

Calcium ion-dependent phosphorylation of human erythrocyte membranes.

Calcium ions promote the rapid transfer of the terminal phosphate of ATP to a protein of human erythrocyte membranes. The concentration of Ca2+ for half-maximal effect is 7 muM. At nonlimiting ATP concentrations the level of 32P incorporated by the membranes is independent of the presence or absence of Mg2+. The number of phosphorylating sites in a single erythrocyte membrane is about 700. The influence of pH on the rate of hydrolysis of the bound phosphate and its rapid release on exposure to hydroxylamine are both consistent with an acylphosphate bond. The phosphate in the protein undergoes rapid turnover. Enzymatic splitting of the phosphate is stimulated by Mg2+ but not by Ca2+. It is proposed that Mg2+ accelerates the splitting of the phosphate by favoring the conversion of the phosphoprotein from a state of low reactivity to a state of high reactivity towards water. The reactions described probably are intermediate steps in the hydrolysis of ATP catalyzed by the Ca2+-dependent ATPase of human erythrocyte membranes.     

Calcium binding by the erythrocyte membrane

Calcium binding to isolated erythrocyte membranes was stimulated by ATP. This stimulatory effect of ATP required Mg²⁺.Ethacrynic acid and ruthenium red inhibited the stimulatory effect of ATP.About 80% of the bound Ca²⁺ was associated with the membrane protein.The strongly bound Ca²⁺ was confined to two high molecular weight membrane proteins.Increasing amounts of Ca²⁺ bound to the membrane inhibited Na⁺ binding in the presence of ATP.     

Calmodulin binding proteins in human erythrocyte membranes

Human erythrocyte membranes reveal different calmodulin-binding proteins determined by a 125I-calmodulin gel overlay procedure. Beside the well-established Ca2+-transport ATPase, other proteins (205, 91, 72 and 42 kDa) bind calmodulin in a Ca2+-dependent manner. Two proteins of the human erythrocyte membrane are able to bind calmodulin only in the absence of Ca2+. One of them (76 kDa) is probably an integral, the other (240 kDa) a peripheral protein.     

Muscarinic cholinergic binding in human erythrocyte membranes

Human erythrocyte ghosts contain a small population of muscarinic cholinergic receptors, as evidenced by their high affinity binding of radiolabeled quinuclinidinyl benzilate ([³H]QNB). The apparent K_D is 1.3 × 10^?9 M and the receptor sites are saturated at a QNB concentration of 5 nM. The number of sites is 23 fmoles/mg membrane protein. The pharmacological profile of the specific binding is similar to that of neural membranes. The binding is not stereoselective for the d and 1 isomers of QNB, a situation which prevails in the muscarinic receptors of another peripheral cholinergic system, the rat iris, but not in the central nervous system.     

Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes.

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest ¹²⁵I-labelled casein, covalently linked to latex beads, have been examined.Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8–9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade ¹²⁵I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Glucose- and phlorrhizin-protected thiol groups in pig intestinal brush-border membranes.

Purified brush borders, prepared fro newborn pig intestine, were incubated in the presence of 203Hg-labelled p-chloromercuribenzenesulphonic acid and the membrane proteins later separated by polyacrylamide-gel electrophoresis. The presence of either D-glucose or phlorrhizin, during a preliminary incubation in non-radioactive p-chloromercuribenzenesulphonic acid, increased the subsequent binding of the 203Hg-labelled compound to a protein of molecular weight 31500. This increase appeared to be specific for the low-molecular-weight protein, provided that the concentration of protecting agent used corresponded to that used to produce a biological response in the intact tissue. These results are discussed in relation to the known properties of other presumptive sugar carriers isolated from different membranes.     

Calcium binding by smooth muscle plasma membranes

Calcium binding by the vesiculate fraction of rabbit small intestine myocyte plasma membranes was studied. It was shown that the membrane fraction as well as the muscle tissue contain two types of Ca2(+)-binding sites with binding constants of 2.3-2.5 x 10(4) and 2.1-1.25 x 10(3) M-1. The number of binding sites and their affinity for Ca2+ depend on the presence in the incubation medium of Mg2+, Na+ and ATP.     

Calcium ion binding by thymocyte plasma membranes

The equilibrium in calcium ions binding to isolated cattle thymocyte plasma membranes within the concentration range 1.10(-5)--5.10(-4) M has been investigated. The plasma membranes has been shown to possess two kinds of Ca(2+)-binding sites with the association constants 2,35.10(5) and 7.64.10(2) M-1 and numbers of binding sites 3.45.10(-4) and 4.55.10(-3) mole.g-1 protein respectively.