A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.     

Cold-labile hemolysin produced by limited proteolysis of theta-toxin from Clostridium perfringens

A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin. The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity. C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C. At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin. It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis. The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained.     

Effect of lipidic factors on membrane cholesterol topology--mode of binding of theta-toxin to cholesterol in liposomes.

We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglycerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14-18 carbon-hydrocarbon chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for theta-toxin.     

A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region.

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.     

Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites.

Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 X 10(-20) mol/cell per s at 25 degrees C compared with 254 X 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).     

Two subpopulations of alpha 1-adrenergic receptors with high and low affinity for agonists: short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including beta-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in alpha 1-adrenergic receptors induced by agonists. alpha 1-receptors were studied on DDT1 MF-2 smooth muscle cells (DDT1-MF-2 cells) by specific [3H]prazosin binding. In competition binding on membranes and on intact cells at 4 degrees C or at 37 degrees C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37 degrees C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [3H]prazosin binding inhibited by 20 microM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [3H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4 degrees C, but only 30% at 37 degrees C. On DDT1-MF-2 cells preequilibrated with [3H]prazosin at 4 degrees C, and then shifted to 37 degrees C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4 degrees C it is the native form of alpha 1-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37 degrees C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 microM norepinephrine. The nature of this low-affinity form was further investigated. On DDT1-MF-2 cells preincubated with the agonist and then extensively washed at 4 degrees C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [3H]prazosin at 4 degrees C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4 degrees C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37 degrees C may be due to the agonist-induced sequestration of alpha 1-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Similarities between complement-mediated and streptolysin S-mediated hemolysis

The oxygen-stable hemolysin streptolysin S (SLS) of Streptococcus pyogenes is encoded in part by the pel/sagA gene product. Antibodies to a synthetic peptide from the C terminus of the Pel/SagA open reading frame inhibited hemolysis mediated by both culture supernatants from multiple M serotypes of S. pyogenes isolates or a commercially available SLS preparation. Analysis of the SLS-mediated hemolytic reaction demonstrated that it was temperature- and concentration-dependent. Like complement-mediated hemolysis it conforms to the prediction of a one-hit mechanism of hemolysis. A number of intermediates in the SLS-mediated hemolysis of sheep erythrocytes could be distinguished. SLS could bind to erythrocytes below 17 degrees C; however, lysis could only occur at temperatures >23 degrees C. Following binding of SLS and washing, a papain-sensitive intermediate could be distinguished prior to insertion of the SLS complex into the erythrocyte membrane, which resulted in formation of a transmembrane pore and led to irreversible osmotic lysis of the cell. These intermediates were similar to those described previously during complement-mediated hemolysis.     

Internalization of basic fibroblast growth factor by Chinese hamster lung fibroblast cells: involvement of several pathways.

Subconfluent Chinese hamster lung fibroblast cells (CCL39) which express high- and low-affinity binding sites for basic fibroblast growth factor (bFGF) were used to study bFGF internalization. Kinetics at 37 degrees C indicated that this process was complex and involved various pathways with regard to the ligand concentration used. Internalization with 6 to 45 pM of 125I-r-bFGF led to a steady state that lasted up to 3 h without any appearance of 125I-labeled degradation products in the cell-culture medium, suggesting that the endocytosis reached equilibrium. Furthermore, binding data at steady state, at 37 degrees C, revealed a two-phase Scatchard curve suggesting the involvement of two families of interaction sites in the process of internalization. Apparent dissociation constants were estimated to be 20 pM and 58 nM, respectively, and the number of bFGF molecules involved per cell, 4300 and 1.3 x 10(6), respectively. These data were in good agreement with those obtained from binding experiments at equilibrium at 4 degrees C. Besides, higher concentrations of 125I-r-bFGF (greater than 47 pM) induced an internalization process which did not reach steady state and was not saturable. These results suggest that CCL39 cells could internalize bFGF by various pathways involving high- and low-affinity binding sites.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens alpha-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.     

Temperature effect on subclasses of muscarinic receptors in rat colon, heart and cerebral cortex

The high-affinity muscarinic antagonist /3H/-Quinuclidinyl benzilate (/3H/-QNB) has been used to label muscarinic receptors in a crude membrane fraction of rat cerebral cortex, colon and heart. The inhibition of /3H/-QNB binding by Atropine, Oxotremorine and Pirenzepine was investigated at three temperatures: 37 degrees C, 22 degrees C and 10 degrees C. The IC50 values and the proportion of high (Rt1) and low (Rt2) affinity binding sites were determined for the three compounds. When the temperature were lowered from 37 degrees C to 10 degrees C, in the agonist and antagonist dissociation constants decreased in all tissues. Changes in temperature did not modify Rt1 or Rt2 values for Oxotremorine and Pirenzepine. The results show marked temperature-dependent modifications of IC50 values for muscarinic receptors of high- and low-affinity sites in rat cerebral cortex, colon or heart.     

Solubilization and assay of a colony-stimulating factor receptor from murine macrophages

The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).     

Complement inhibitor(s) released by leukocytes. III. Evidence for a "new" C1 inhibitor in the supernatants of short-term cultures of mouse spleen and thymus cells.

Mouse spleen or thymus cells in short-term culture release a factor, designated S, that binds to sheep erythrocytes (E). Supernatant-treated sheep erythrocytes (SE) are capable of fixing and transferring the activated first component of guinea pig complement. SEC1, however is not capable of initiating hemolysis by the rest of the complement components. SE is capable of binding but not activating native C1; native C1 bound to SE seems irreversibly inhibited. Evidence is presented that S may be the same factor as the previously described inhibitor released by mouse spleen or thymus cells that inhibits the utilization of C2 by EAC14.     

Effects of temperature and cholesterol on human erythrocyte membranes

The effects of temperature and cholesterol on the membrane fluidity of human erythrocytes were studied using 5-nitroxide stearic acid (5NS), 12-nitroxide stearic acid (12NS), and 16-nitroxide stearic acid (16NS). Human erythrocytes and their lipid vesicles were treated in the range of 5--55 degrees C. In erythrocytes, ESR signals for 12NS and 16NS showed line broadening above 40 degrees C, whereas those for 5NS became sharper with increasing temperature as was the case with the signals of lipid vesicles for each label molecule. Lipid extraction from the heated sample caused no radical reduction. Only in 12NS-labeled erythrocytes did a weakly immobilized component and a strongly immobilized component appear. In the time course at 50 degrees C, the former decreased and the latter remained constant. From the ratio of both components, it was found that the interaction of the label molecules with the binding sites was determined by the physical state of the membrane. Furthermore, the dependence on temperature of the molecular motion of the labels in the cell membrane was irreversible above 40 degrees C. On addition of cholesterol to the membrane, the outer hyperfine splittings for 12NS and 16NS increased but that for 5NS decreased at C/P greater than 1, perhaps indicating a spread between the head groups of phospholipids by cholesterol.     

Enzymatic oxidation of membrane cholesterol in relation to lysis of sheep erythrocytes by corynebacterial enzymes

Synergistic hemolysis of sheep erythrocytes brought about by the combined action of Corynebacterium ovis (C. pseudotuberculosis) and Corynebacterium equi depends upon the extracellular sphingomyelin-specific phospholipase D of the former species and a partially characterized agent(s) of the latter. Fractionation of the culture supernatant of C. equi revealed a cholesterol oxidase which was purified to near homogeneity by gel filtration and isoelectric focusing. The enzyme was isoelectric at pH 9–10 and had a molecular weight of 61,000. Sheep erythrocytes pretreated with purified sphingomyelinase D of C. ovis were hemolyzed by incubation with C. equi cholesterol oxidase or by the same enzyme from Brevibacterium sp. Lipid analysis revealed complete conversion of membrane cholesterol to cholest-4-en-3-one, the product of cholesterol oxidase action. Cells not pretreated with sphingomyelinase D did not undergo cholesterol oxidation or hemolysis when treated with cholesterol oxidase. Studies with crude culture supernatant of C. equi confirmed the presence of a phospholipase active in hydrolyzing ceramide phosphate generated in the erythrocyte membrane by C. ovis sphingomyelinase. Ceramide thus produced in the membrane is known to make the cells labile to hemolysis. There are, therefore, at least two mechanisms underlying synergistic hemolysis by these coryne-bacteria.     

C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin.

Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.     

The interaction of retinol-binding protein with its plasma-membrane receptor.

125I-labelled retinol-binding protein (RBP) bound to specific receptors in human placental brush-border membranes. Binding at 22 degrees C reached equilibrium within 15 min, but prolonged incubation caused a subsequent decline. Scatchard analysis of the equilibrium binding data at 22 degrees C and 15 min showed high-(3.0 +/- 2.7 x 10(-9) M) and low-(9.5 +/- 3.5 x 10(-8) M) affinity binding components. 125I-RBP, bound to membranes at 22 degrees C for 15 min and subsequently dissociated with excess unlabelled RBP, exhibited biphasic dissociation kinetics consisting of fast and slow components of release. In contrast, Scatchard analysis and dissociation kinetics of the binding that had taken place at 37 degrees C for 1 h showed the fast-dissociating/low-affinity binding component, but little of the slow-dissociating/higher-affinity binding component. When 125I-RBP, after incubation with membranes at 37 degrees C for 1 h, was re-isolated and subjected to dissociation kinetic analysis using a fresh batch of membranes, the fast-dissociating phase was unchanged, but the slow phase was almost absent. The complex kinetics were interpreted in terms of a heterogeneity in RBP consisting of high- and low-affinity binding forms. The higher-affinity-binding form is thought to be converted into the lower-affinity state on binding to the receptor. Transthyretin inhibited 125I-RBP binding to the membrane, suggesting that free, rather than transthyretin-associated, RBP bound to the receptor. The RBP receptor was trypsin-, heat- and thiol-group-specific-reagent sensitive and was highly specific for RBP.     

Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes

Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.     

Vicia graminea lectin binding to human M and N erythrocytes

The mode of binding of Vicia graminea¹²⁵I-labelled lectin to human M and N erythrocytes at 4°C has been investigated. The labelled lectin retained the full activity of native lectin. Lectin association at 4°C was characterized by a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 3 to 5 min, reaching steady-state within 15 min. Incubation of cells for 15 min at 4°C with increasing concentrations of Vicia graminea¹²⁵I-labelled lectin showed that saturation binding occurred. Scatchard analysis of equilibrium data determined over a wide range of lectin concentrations yielded a curvilinear plot with an upward concave slope; this representation indicated that there was not a single homogeneous class of noninteracting binding sites. This result could indicate two or more independent classes of binding sites or one class of interacting sites exhibiting negative cooperativity. Since unlabelled lectin, which at the concentration used, rapidly binds to available receptors, did not affect the dissociation rate of the labelled lectin and since identical Scatchard plots were found using native and formaldehyde-fixed erythrocytes we conclude that there are two classes of independent Vicia graminea binding sites on human erythrocytes. Computer analysis of the Scatchard plots gave high- and low-affinity constant (7.07±1.1) · 10⁷ M^?1 and (0.2±0.01) · 10⁷ M^?1, respectively, for N erythrocytes and (1.13±0.18) · 10⁷ M^?1 and (0.24±0.01) · 10⁷ M^?1, respectively for the M cells. N erythrocytes were estimated to have 0.085 · 10⁵ high-affinity and 2.1 · 10⁵ low-affinity sites and M erythrocytes, 0.011 · 10⁵ high affinity and 0.13 · 10⁵ low-affinity sites. N cells therefore have 10-times as many sites as M cells. Studies of the dissociation of ¹²⁵I-labelled lectin from N and M cells in the presence of unlabelled lectin gave dissociation rate constants of 51 · 10^?4 s^?1 and 1.97 · 10^?4 s^?1 for the high- and low-affinity sites of N cells and 13 · 10^?4 s^?1 and 1.6 · 10^?4 s^?1 for the high- and low-affinitym sites of M cells, indicating that the binding of Vicia graminea lectin to human erythrocytes is reversible.