Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

Ceruloplasmin receptor on human erythrocytes

The structural fragments of the human ceruloplasmin (CP) molecule and of erythrocyte receptors which provide for the specific interaction of CP with erythrocytes were identified, and their properties were investigated. The interaction of CP with erythrocytes, both intact and treated with neuroaminidase and proteolytic enzymes (trypsin, chymotrypsin, papaine, pronase E) is described. Experiments with CP reception were performed at 4 degrees C, using [125I]CP and [125I]asialo-CP. The parameters of binding were determined in Scatchard plots. It was demonstrated that the specific binding of CP to erythrocyte receptors is determined by its interaction with two structural sites of the carbohydrate moiety of the CP molecule, i.e., the terminal residues of sialic acids and a site, (formula; see text) located at a large distance from the chain terminus.     

Explanation of the pseudohypoaldosteronism (PHA)-stress syndrome with an artificial aldosterone receptor model

A receptor for aldosterone was studied in the cytosol of rectal mucosa of two sisters (M.A., M.B.) with the clinical manifestations of pseudohypoaldosteronism (PHA). Compared to age matched controls the patients showed a decreased affinity for aldosterone (M.A. Kd1: 0.18 nM, Kd2: 4.55 nM; Nmax1: 0.185 fmol/mg cytosol protein (CP), Nmax2: 3.12 fmol/mg CP, respectively). In an attempt to find an explanation for the phenomenon of stress-induced electrolyte imbalance in PHA patients an experimental set up was designed, using aldosterone antibody material as artificial aldosterone receptor. Specific binding was evaluated in addition with and without a 25-100-fold molar excess of dexamethasone (DEX) in order to overcome the glucocorticoid affinity of the aldosterone receptor, a phenomenon proposed to be the cause for the severe consequences of stress in some patients with PHA. The aldosterone antiserum showed two binding sites, similar to the natural receptor (Kd1: 0.15 nM, Kd2: 1.30 nM; Nmax: 30 fmol/mg CP and 130 fmol/mg CP, respectively). Under the influence of DEX the high affinity binding site (Kd1) was occupied by the glucocorticoidanalogon (Kd: 1.30 nM; Nmax: 125 fmol/mg CP). In conclusion, in stress situations, with increased quantities of glucocorticoid circulating, the high affinity binding site of the aldosterone receptor might be occupied by the glucocorticoids, while the low affinity binding site in PHA patients might not have sufficient binding capacity to maintain the electrolyte balance.     

Variations on fibrinogen-erythrocyte interactions during cell aging

Erythrocyte hyperaggregation, a cardiovascular risk factor, is considered to be caused by an increase in plasma adhesion proteins, particularly fibrinogen. We have recently reported a specific binding between fibrinogen and an erythrocyte integrin receptor with a β(3) or β(3)-like subunit. In this study we evaluate the influence of erythrocyte aging on the fibrinogen binding. By atomic force microscopy-based force spectroscopy measurements we found that increasing erythrocyte age, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence but not its force. This observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. We conclude that upon erythrocyte aging the number of fibrinogen molecules bound to each cell decreases significantly, due to the progressive impairment of the specific fibrinogen-erythrocyte receptor interaction. Knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen content in blood, which could disturb the blood flow. Our data also show that the sialic acids exposed on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating its binding to the erythrocyte membrane receptor.     

Studies on receptor interaction of ceruloplasmin with human red blood cells

The interaction of CP with the red blood cell (RBC) receptor was shown to be a Ca2+ dependent process and be limited by CP binding on RBC membrane which is not followed by CP transport through the membrane into RBC. The nature of receptor interaction was determined. It was shown that receptors are formed by glycoproteins of PAS1 and PAS2 (glycoforin dimer and monomer, respectively) and terminal residues of sialic acid of these glycoproteins are important for CP reception. Receptor carbohydrate specificity was determined. Biantennary structure of CP molecule carbohydrate moiety which is bound to the receptor owing to 2 structural fragments: sialic acid terminal residues and the fragment including acetylglucosamine dimer and fucose, plays the main role in CP reception.     

Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component.

We have recently reported that the highly potent beta-adrenergic affinity label [125I]bromoacetylamino cyanopindolol ([125I]BAM-CYP) irreversibly blocks the turkey erythrocyte beta-adrenoceptor binding site by combining with a receptor-associated non-protein component. In this communication, we report: lipid labelling is inhibited by beta 1-adrenergic ligands with the potency ratio and stereospecificity characteristic for the turkey erythrocyte beta 1-adrenoceptor; the tagged component is a glycolipid, probably a ganglioside; [125I]BAM-CYP-blocked receptor, after solubilization in deoxycholate, can be separated from the [125I]BAM-CYP-glycolipid with restoration of the binding capacity of the beta 1-adrenoceptor protein; the tightly associated [125I]BAM-CYP-labelled glycolipid can be displaced by a glycolipid mixture extracted from turkey erythrocyte membranes but not by bovine brain gangliosides, when the blocked receptor is solubilized in digitonin. This is the first direct demonstration that a receptor protein is associated with a specific membrane lipid. The possibility that glycolipids play a role in receptor-mediated signal transduction is discussed in view of these findings and in view of data from the literature.     

Identification of adenylate cyclase-coupled beta-adrenergic receptors in frog erythrocytes with (minus)-[3-H] alprenolol.

(minus)-Alprenolol, a potent, competitive beta-adrenergic antagonist labeled to high specific activity with tritium (17 Ci per mmol), has been used to identify binding sites in frog erythrocyte membranes having many of the characteristics to be expected of the beta-adrenergic receptors which are linked to adenylate cyclase in these membranes. The chromatographic behavior and biological activity of the labeled and native drug were essentially identical. (minus)-Alprenolol and (minus)-[3-H]alprenolol both competitively antagonize isoproterenol stimulation of frog erythrocyte membrane adenylate cyclase with a KD OF 5 TO 10 NM. (minus)-[3-H]Alprenolol binding to sites in the frog erythrocyte membranes was studied by a centrifugal assay. At 37 degrees, equilibrium binding was established within 5 min and the half-time for dissociation of bound (minus)-[3-H]alprenolol was approximately 30 s. This rapid onset and dissociation of (minus)-[3-H]alprenolol binding was in good agreement with the rapid onset of action of beta-adrenergic agonists and antagonists on the frog erythrocyte adenylate cyclase. (minus)-[3-H]Alprenolol binding was saturable. There were 0.25 to 0.35 pmol of (minus)-[3-H]alprenolol binding sites per mg of protein corresponding to 1300 to 1800 binding sites per intact frog erythrocyte. The binding sites showed half-maximal saturation at 5.0 to 10 nM (minus)-[3-H]alprenolol, which is in good agreement with the KD for alprenolol antagonism of isoproterenol stimulation of adenylate cyclase. The (minus)-[3-H]alprenolol binding sites exhibited strict stereospecificity. (minus)-Stereoisomers of beta-adrenergic antagonists or agonists were approximately 2 orders of magnitude more potent than the (+)-stereoisomers in competing for the binding sites. Comparable stereospecificity was apparent when agonists and antagonists were tested for their ability to interact with the adenylate cyclase-coupled beta-adrenergic receptors in the membranes. Potency series of 11 agonists and 13 antagonists for inhibition of binding and interaction with adenylate cyclase were identical and were characteristic of a beta2-adrenergic receptor. A variety of nonphysiologically active compounds containing a catechol moiety as well as several metabolites and cholinergic agents did not inhibit (minus)-[3-H]alprenolol binding or interact significantly as agonists or antagonists with the adenylate cyclase. The (minus)-[3-H]alprenolol binding sites studied appear to be equivalent to the beta-adrenergic receptor binding sites in the frog erythrocyte membranes.     

Various properties of the ceruloplasmin receptor, isolated from human erythrocyte membranes

An electrophoretically pure preparation of ceruloplasmin (CP) receptor which retains its ability to bind to CP was isolated from human erythrocyte membranes. It was found that in terms of molecular mass, number and size of spontaneously proteolytic fragments as well as antigenicity, the CP receptor molecule strongly resembles that of CP. A comparative analysis of two-dimensional peptide maps of full tryptic digests of the both protein revealed that about 30% of CP peptides are identical in respect of their electrophoretic and chromatographic mobilities which points to the genetic independence of these proteins. The roles of CP and CP receptor in copper metabolism are discussed.     

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) interacts with the band 3 receptor to promote erythrocyte invasion by malaria parasites

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) is an erythrocyte binding protein known to be involved in malarial parasite invasion. Although anti-GAMA antibodies have been shown to block GAMA attachment to the erythrocyte surface and subsequently inhibit parasite invasion, little is known about the molecular mechanisms by which GAMA promotes the invasion process. In this study, LC-MS analysis was performed on the erythrocyte membrane to identify the specific receptor that interacts with GAMA. We found that ankyrin 1 and the band 3 membrane protein showed affinity for GAMA, and characterization of their binding specificity indicated that both Plasmodium falciparum and Plasmodium vivax GAMA bound to the same extracellular loop of band 3 (loop 5). In addition, we show the interaction between GAMA and band 3 was sensitive to chymotrypsin. Furthermore, antibodies against band 3 loop 5 were able to reduce the binding activity of GAMA to erythrocytes and inhibit the invasion of P. falciparum merozoites into human erythrocytes, whereas antibodies against P. falciparum GAMA (PfGAMA)-Tr3 only slightly reduced P. falciparum invasion. The identification and characterization of the erythrocyte GAMA receptor is a novel finding that identifies an essential mechanism of parasite invasion of host erythrocytes.     

Antioxidant enzymes and lipid peroxides in children with cerebral palsy

Impaired antioxidant mechanisms are unable to inactivate free radicals that may induce a number of pathophysiological processes and result in cell injury. Thus, any abnormality in antioxidant defense systems could affect neurodevelopmental processes and could have an important role in the etiology of cerebral palsy (CP). The plasma levels of lipid peroxidation as plasma levels of malondialdehyde (MDA), activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) in plasma and erythrocytes were investigated in 34 CP children and compared with 61 normal controls. SOD, GPx and GR activities were spectrophotometrically assayed. Activities of SOD, GPx and GR in plasma did not differ significantly between CP children and the control group. Activities of erythrocyte GR in the CP patients were significantly lower compared with controls. MDA concentration did not differ statistically between the CP children and healthy subjects. In conclusion our results suggest that increased activities of erythrocyte GPx and decreased erythrocyte GR activities might be due to lesser physical activity of children with CP.     

Demonstration of corticotropin-releasing factor binding sites on human and rat erythrocyte membranes and their modulation by chronic ethanol treatment in rats

In a previous study we reported the presence of specific corticotropin-releasing factor (CRF) binding sites in peripheral tissues of the rat (Endocrinology, 116, 2152, 1985). Using 125I-labeled rat or human CRF, specific CRF binding sites were identified on rat and human erythrocytes, but not on lymphocytes or platelets. Furthermore, identical CRF binding was observed in the presence of intact erythrocytes or lysed erythrocyte membranes. Maximal binding of 125I-CRF occurred within 25 min at 4 degrees C and was saturable. Scatchard analysis of CRF binding to erythrocyte membranes revealed the existence of a single class of binding site. Chronic exposure of rats to ethanol vapor, known to lower specific CRF binding to pituitary tissue by 35%, also decreased 125I-rat CRF binding to erythrocyte membranes by approximately 45%, which was due to a decrease in the number of CRF binding sites. The parallel decrease of CRF binding to rat-erythrocyte and pituitary membranes following chronic ethanol treatment suggests that CRF binding to erythrocyte and pituitary membranes is modulated in a similar direction, which further suggests that the determination of CRF binding to erythrocytes may provide an important clinical tool to indirectly assess CRF-receptor levels in the pituitary gland and thereby enhance our understanding of ethanol-induced disorders of the hypothalamic-pituitary-adrenal axis in patients.     

The oligosaccharide moiety of the beta 1-adrenergic receptor from turkey erythrocytes has a biantennary, N-acetyllactosamine-containing structure

The turkey erythrocyte beta 1-adrenergic receptor can be purified by affinity chromatography on alprenolol-Sepharose and characterized by photoaffinity labeling with N-(p-azido-m-[125I]iodobenzyl)-carazolol. Through the use of the specific glycosidases neuraminidase and endo-beta-N-acetylglucosaminidase H and affinity chromatography on lectin-Sepharose gels, we show here that the receptor is an N-glycosyl protein that contains complex carbohydrate chains. No high-mannose carbohydrate chains appear to be present. The binding of the radiolabeled antagonist dihydroalprenolol to the receptor is affected neither by the enzymic treatments nor by the presence of lectins, suggesting that the carbohydrate moiety is not involved in the catecholamine binding site.     

Aberrant porphyrin metabolism in hepatocellular carcinoma

In 15 patients with hepatocellular carcinoma (HCC) and 14 patients with liver cirrhosis (LC), urinary excretions of delta-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrin (UP), coproporphyrin (CP), and erythrocyte contents of CP and protoporphyrin (PP) were examined. In patients with HCC, urinary excretions of ALA and PBG and erythrocyte contents of CP and PP were not increased, but urinary excretions of UP and CP were significantly increased more than those of LC patients. Urinary excretions of UP and CP had no correlations with liver function tests and excretion of UP correlated slightly with blood hemoglobin level. After administration of ALA intravenously, urinary excretions of UP and CP were clearly increased in patients with HCC compared to normal controls. A Red fluorescent area was present at the cancerous area but not in the noncancerous cirrhotic area in a patient with HCC. These results suggest that aberrant porphyrin metabolism occurred in patients with HCC compared to other liver diseases.     

Antipeptide antibodies that recognize a lymphocyte substance P receptor

In an effort to investigate the presence of substance P (SP) receptors on lymphocytes, polyclonal antibodies against SP receptors were developed. The immunogen used to generate these antibodies was a peptide encoded by an RNA complementary to the mRNA for SP. The rationale for using this SP complementary peptide (termed SP CP) as an immunogen resulted from the observation that 3H-SP bound to microtiter wells coated with SP CP in a dose dependent and saturable fashion. Furthermore, binding was blocked with excess unlabeled SP or SP antagonist, D-Pro2-D-Phe7-D-Trp9-SP. Inasmuch as the peptide, SP CP, specifically bound 3H-SP, we hypothesized that antibodies against this peptide might recognize a SP receptor binding site. Using the SP receptor positive lymphoblast cell line, IM-9, affinity-purified antibodies against SP CP but not antibodies against keyhole limpet hemocyanin recognized a molecule on the surface of IM-9 cells. Anti-SP CP binding to IM-9 cells was blocked with excess SP antagonist, suggesting that the antibody and the SP antagonist were competing for the same binding site. In support of this possibility, anti-SP CP antibodies blocked 3H-SP binding to IM-9 cells. An immunoaffinity column coupled with antibodies against SP CP bound protein from solubilized IM-9 cells. This isolated protein bound 125I-Tyr8-SP and binding was specifically blocked with SP as well as by SP antagonist, neurokinin A, and eledoisin. Passthrough material did not bind SP suggesting that a SP receptor had been purified. Western blot analysis of solubilized IM-9 cell proteins using anti-SP CP antibodies but not preimmune IgG recognized a single protein of 58,000 D. Taken together, these results demonstrate that antibodies against SP CP recognize a SP receptor present on the lymphocyte cell line, IM-9.     

The erythrocyte insulin receptor response to insulin induced hypoglycaemia

The response of the erythrocyte insulin receptor to a prolonged intravenous infusion of insulin has been measured in normal individuals during hypoglycaemia and when hypoglycaemia was prevented by the concurrent infusion of glucose. When euglycaemia was maintained, mean (+/- S.D.) specific insulin binding following the 5 hour insulin infusion was unchanged (6.9 +/- 2.1 to 6.65 +/- 2.2% bound per 2.25 X 10(9) erythrocytes). In the presence of mild hypoglycaemia, mean (+/- SD) specific insulin binding rose from 6.6 +/- 2.3 to 7.6 +/- 2.5% bound per 2.25 X 10(9) erythrocytes (P less than 0.01), after 5 hours. This increase was due to increased receptor affinity. It was not correlated with the increase in the concentration of any individual counter-regulatory hormone. Initial insulin receptor binding correlated strongly with the subsequent decline in plasma glucose concentration (r = 0.9527; P less than 0.01). Thus, acute hyperinsulinaemia, when associated with hypoglycaemia, does not result in downregulation of insulin receptors on erythrocytes but rather results in increased receptor binding. Consequently, the insulin receptor may not play an active role in protecting the individual against acute hypoglycaemia.     

Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus

Viruses use sophisticated mechanisms to allow the specific packaging of their genome over that of host nucleic acids. We examined the in vitro assembly of the Cowpea chlorotic mottle virus (CCMV) and observed that assembly with viral RNA follows two different mechanisms. Initially, CCMV capsid protein (CP) dimers bind RNA with low cooperativity and form virus-like particles of 90 CP dimers and one copy of RNA. Longer incubation reveals a different assembly path. At a stoichiometry of about ten CP dimers per RNA, the CP slowly folds the RNA into a compact structure that can be bound with high cooperativity by additional CP dimers. This folding process is exclusively a function of CP quaternary structure and is independent of RNA sequence. CP-induced folding is distinct from RNA folding that depends on base-pairing to stabilize tertiary structure. We hypothesize that specific encapsidation of viral RNA is a three-step process: specific binding by a few copies of CP, RNA folding, and then cooperative binding of CP to the "labeled" nucleoprotein complex. This mechanism, observed in a plant virus, may be applicable to other viruses that do not halt synthesis of host nucleic acid, including HIV.     

Dietary trans fatty acids modulate erythrocyte membrane fatty acyl composition and insulin binding in monkeys

The substitution of trans- for half of the cis-monounsaturated fatty acids in the diet of Macaca fasicularis monkeys resulted in alterations in erythrocyte fatty acid composition and insulin receptor properties but not in membrane fluidity. Both cis and trans diets contained 10% fat and similar fatty acid compositions, except that approximately 50% of the cis-octadecenoate (c-18:1) in the cis diet was replaced with trans-octadecenoate isomers (t-18:1) in the trans diet. Compared with the cis diet, the trans diet resulted in the incorporation of approximately 11% t-18:1, an approximately 50% decrease in c-18:1, an approximately 16% decrease in total saturated fatty acids, and an approximately 20% increase in 18:2(n-6) in erythrocyte membrane lipids. The increase in 18:2(n-6) may reflect on homeostatic mechanisms designed to maintain overall membrane fluidity, as no diet-related changes in fluidity were observed with diphenylhexatriene steady state fluorescence polarization. Values observed for insulin binding and insulin receptor number were higher and binding affinity was lower in monkeys fed the cis diet. In the absence of an effect on overall membrane fluidity, altered receptor activity suggests that insulin receptor activity is dynamic, requiring specific fluid membrane subdomains or highly specific fatty acid-protein interactions.     

Erythrocyte insulin receptor characteristics and erythrocyte membrane lipid composition in healthy men

The cell membrane plays an important role in the mechanism of insulin action. To test whether erythrocyte insulin receptor characteristics are related to the erythrocyte membrane lipid composition, 11 healthy volunteers were studied. The relationship between insulin binding to erythrocytes, the number of receptors per cell and the affinity of receptors to insulin on the one hand and total phospholipid fatty acid (FA) composition and cholesterol/phospholipid molar ratio in the erythrocyte membrane on the other hand were evaluated. 1. We found a significant negative correlation between specific insulin binding and the proportion of n-6 essential FA in erythrocyte membrane phospholipids, especially linoleic acid (r = -0.82, p less than 0.01) and arachidonic acid (r = -0.73, p less than 0.05). On the other hand, a significant positive correlation between insulin binding and the proportion of nonessential FA (r = +0.65, p less than 0.05) was seen. Number of receptors per cell and the affinity of receptors were not significantly related to phospholipid FA composition. 2. There was no significant correlation between insulin receptor characteristics and the cholesterol/phospholipid molar ratio in the erythrocyte membrane. The data presented support the hypothesis that the FA pattern of membrane total phospholipids may modify the properties of insulin receptors.     

Identification of an elicitor active site within the three-dimensional structure of the tobacco mosaic tobamovirus coat protein.

The coat protein (CP) of tobacco mosaic tobamovirus (TMV) elicits the hypersensitive response (HR) conferred by the N' gene from Nicotiana sylvestris. This study presents evidence demonstrating a critical role for a specific CP structural site in eliciting this HR. Based on the known structure of the TMV CP, specific substitutions were created within the CP of the elicitor strain P20L to identify structural areas essential for host recognition. Of 32 substitutions made, 14 conferred either a temperature-sensitive (loss of the HR at 29 degrees C) or a knockout (loss of the HR at 25 degrees C) HR phenotype in N.sylvestris. These essential residues were noncontiguous in position; however, within the three-dimensional CP structure, all resided primarily along the right face of the molecule's helical bundle. Substitutions that did not affect the HR phenotype either were located outside of this area or were conservative in change. In addition, placing two temperature-sensitive substitutions within the same CP resulted in lowering temperature sensitivity from 29 to 27 degrees C. This additive effect suggests that residues essential for HR elicitation contribute independently to host recognition. This feature is characteristic of recognition surfaces. The presence of a specific elicitor active site within the three-dimensional structure of the TMV CP is consistent with binding of a host-encoded receptor and demonstrates the importance of CP structure in HR specificity.     

Comparison of the protective effects on the erythrocytes of ceruloplasmin from the blood of healthy donors and patients with hepatocerebral dystrophy

The binding of the ceruloplasmin (CP) from the healthy donor's blood and of ceruloplasmin--like protein (p-CP) isolated from the Wilson disease patient's blood with erythrocytes (RBC) of healthy donors and with RBC of Wilson's patients (p-RBC) was investigated. It was shown, that the CP number of binding sites both on the RBC and p-RBC was significantly lower than that for p-CP, but Kd value for p-CP binding of the both types of erythrocytes was approximately 10 times higher than Kd value for CP. The protective action of CP on copper stimulated hemolysis is almost 3 times higher than that of p-CP. The protective action of CP on ferrous ion stimulated hemolysis doesn't correlate with its ferroxidase activity. On the contrary the protective effect of p-CP which has no ferroxidase activity is more powerful than that of CP.