The oligomeric state of spectrin in the rat erythrocyte membrane skeleton

The oligomeric state of spectrin in the erythrocyte membrane skeleton of the rat was investigated following extraction in a low ionic strength buffer for 24 and 96 h. All analyses were quantitatively compared with preparations from human erythrocyte membranes. After nondenaturing agarose-polyacrylamide gel electrophoresis, the human samples revealed their characteristic spectrin oligomer pattern; there were high molecular weight complexes near the origin of the gel, followed by several high order oligomers, tetramers, and dimers. The pattern in the rat membrane skeleton also included tetramers and a high molecular weight complex band, but had only one oligomer and no dimers. With time the high molecular weight complex diminished and oligomers accumulated in both the rat and human, while dimers accumulated only in the human and tetramers accumulated only in the rat. Tetramers decreased with time in the human. Extraction of spectrin increased with time and was greater from rat than the human red cell membrane at both time points. The percentage of spectrin and actin in the low ionic strength extract was similar between species, as analyzed by SDS-polyacrylamide electrophoresis, staining, and densitometry. Proteins 4.1 and 4.9 were present in greater percentages in the human. The only temporal effect on monomeric protein composition was an increase of protein A in the rat. There was no species difference in protein A percentage at 24 h, but at 96 h the rat was greater than the human. The results suggest that there are significant differences in the structural arrangement of the rat and human erythrocyte membrane skeleton.     

Species-dependent differences in the influence of ionic strength on potassium transport of erythrocytes. The role of membrane fluidity and Ca2+

The passive Rb+ (K+) efflux from erythrocytes of seven mammalian species was investigated in solutions of physiological and low ionic strength. Furthermore the fluidity of the erythrocyte membrane in the same solutions was estimated by measuring the ESR order parameter. The rate constant of Rb+ (K+) efflux in solution of high ionic strength could be correlated with the order parameter obtained and with the mean number of double bonds to the membrane phospholipid fatty acids. The same relationships could be observed for the low ionic strength solutions if the values for human erythrocytes were excluded. The appearance of Na+, K+, Cl- cotransport to a significant extent, only in human erythrocytes, was supposed to be the reason for this different behaviour of human red blood cells. It was demonstrated that the strong increase of the Rb+ (K+) efflux rate constant for human erythrocytes in low ionic strength solution is not due to Ca2+, as quinine treatment and replacement of all external potassium, both inhibiting the Ca2(+)-induced K+ efflux, did not abolish the increase of (Rb+) K+ efflux in solutions of low ionic strength.     

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.     

Proteolytic self-digestion of bovine erythrocyte membranes

"Self-digestion" of bovine erythrocyte membrane proteins was studied in isolated membrane preparations during prolonged incubation at 37 C. Protease activities associated with the membrane result in progressive degradation of all main erythrocyte membrane proteins, in particular spectrin and Band 3, and formation of lower molecular weight products which have been tentatively assigned to parent molecules. Membrane protein "self-digestion" occurs in a broad pH range (2-11), is inhibited by increased ionic strength and by inhibitors of metalloproteases, cysteine and serine proteases, and activated by low concentrations of SDS. "Self-digestion" also takes place in NaOH-stripped erythrocyte membranes. The activity of a protease involved in the "self-digestion", of apparent molecular weight of about 35,000, was renatured after SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins.     

Relationship of hemolysis buffer structure, pH and ionic strength to spontaneous contour smoothing of isolated erythrocyte membranes

Isolated human erythrocyte membranes crenate when suspended in isotonic medium, but can use MgATP to reduce their net positive curvature, yielding smooth discs and cup forms that eventually undergo endocytosis. An earlier report from this laboratory (Patel, V.P. and Fairbanks, G. (1981) J. Cell Biol. 88, 430-440), has described a phenomenon of ATP-independent shape change in which ghosts prepared by hemolysis and washing in synthetic zwitterionic buffers crenated at 0 degree C, but underwent conversion to smooth discs and cups when warmed in the absence of MgATP. We have further explored the effect of the hemolysis condition on the requirement for ATP in ghost shape change. 25 hemolysis buffers were applied at 10 mM (pH 7.4, 0 degree C). Eight anionic buffers with relatively high ionic strength (e.g., phosphate and diethylmalonic acid (DMA] yielded ghosts requiring ATP for shape change, while two cationic buffers (Bistris and imidazole) and ten synthetic zwitterionic buffers (e.g., Tricine and Hepes) with lower ionic strength produced ghosts that smoothed spontaneously at 30 degrees C. Hemolysis at intermediate ionic strength yielded mixed populations in which spontaneous smoothing was expressed in all-or-none fashion. Maximal ATP-independent shape change was induced by hemolysis at pH 7.3-7.7, while ATP was required after hemolysis at pH less than or equal to 7.1 even when the ionic strength at hemolysis was low. Ghosts requiring ATP could be converted to ATP independence by washing at low ionic strength, but ATP independence could not be reversed readily by washing at high ionic strength. Exposure to low ionic strength at pH greater than 7.1 presumably changes membrane organization in a way that alters the temperature dependence of tensions within the bilayer or skeleton of the composite membrane.     

Procaine as a substrate and possible allosteric effector of cholinesterases

The local anaesthetic procaine showed the properties of an allosteric effector of bovine erythrocyte acetylcholinesterase at low ionic strength; it antagonised inhibition of substrate hydrolysis caused by decamethonium, decreased the rate of ageing of isopropylmethylphosphonyl-acetylcholinesterase, increased the rate of decarbamylation of dimethylcarbamyl-acetylcholinesterase, and interacted synergistically with the nucleophilic alcohol 3,3-dimethyl-1-butanol in the acceleration of decarbamylation. These allosteric effects almost completely disappeared as the ionic strength was increased to a physiological level, and they could not be demonstrated at the physiological ionic strength with membrane-bound human erythrocyte acetylcholinesterase. There was no evidence of significant cooperativity in the binding of procaine to the enzyme, nor in the binding of the substrate acetylthiocholine in the presence of procaine, contrary to reports in the literature for other sources of acetylcholinesterase. Procaine was not hydrolysed by acetylcholinesterase (EC 3.1.1.7) although it is a substrate for serum cholinesterase (EC 3.1.1.8).The possibility that the results at low ionic strength can be explained on the basis of procaine binding to the active site of acetylcholinesterase (at low concentrations) and also to a peripheral allosteric site (at higher concentrations) is discussed. The results confirm the complexity of the kinetics of acetylcholinesterase, and extend the range of compounds with the ability to modify rates of decarbamylation and ageing.     

The interaction of calmodulin with erythrocyte membrane proteins

The method of sedimentation equilibrium in an air-driven ultracentrifuge (Airfuge) has been employed to investigate the interaction of 125I-calmodulin with the cytoskeletal components of the human red cell membrane. The results indicate significant calcium-dependent calmodulin binding activity in the low and high ionic strength extracts of the human erythrocyte membrane. The interaction of 125I-calmodulin with the low ionic strength extract proteins is analysed quantitatively. Further purification of the high ionic strength extract comprising mainly band 2.1 and band 4.1 results in the elution of calmodulin binding activity in a purified fraction of band 4.1.     

Temperature-dependent modes for the binding of the polyene antibiotic amphotericin B to human erythrocyte membranes. A circular dichroism study

The interaction of amphotericin B with isolated human erythrocyte ghosts was monitored by circular dichroism at 37 degrees C and 15 degrees C. Although different, these spectra were not concentration dependent over a concentration range covering the inducement of K+ leakage and hemolysis, which suggests the existence of only one bound amphotericin B species. At 15 degrees C, the spectra indicate that amphotericin B is complexed with membrane cholesterol; the complex formation is saturable but not cooperative. At 37 degrees C new spectra are observed, and their existence is conditioned by the presence of membrane proteins. The binding is cooperative but not saturable. The amphotericin B right side-out vesicles complexation is temperature as well as ionic strength dependent: at high ionic strength it is the same as with ghosts, with the same temperature dependence. At low ionic strength it is characteristic of an interaction with cholesterol, regardless of temperature. In the large unilamellar vesicles reconstituted from the total lipid extracts of erythrocyte membranes, amphotericin B is complexed with cholesterol, regardless of temperature and ionic strength. These results indicate that there are two different modes of amphotericin B complexation with erythrocyte membranes, reversible one in the other, depending on the molecular organization of the membrane and the presence of membrane proteins.     

Recombination of human erythrocyte apoprotein and lipid. I. Interaction of apoprotein and lipid at the air-water interface

The formation and stabilization of a complex between total erythrocyte apoprotein and monolayers of total erythrocyte lipid as measured by changes of surface pressure (Δπ) and rate of change of surface pressure (dπ/dt) was studied as a function of pH, ionic strength, and lipid surface pressure. Penetration of apoprotein into lipid monolayers was favored by conditions in which lipid and apoprotein were oppositely charged. Once the interaction was completed, the resultant surface complex was resistant to large changes in subphase pH and ionic strength as shown by the insensitivity of Δπ to these parameters. The dπ/dt, however, showed strong dependence on pH and ionic strength, but not on lipid surface pressure. A sharp decrease in dπ/dt around pH 3.5–4.5 is associated with the change in apoprotein charge from (+) to (?). Comparison of complex formation between apoprotein and bovine serum albumin, cytochrome c, and human hemoglobin suggests that erythrocyte apoprotein was specialized in its interaction with erythrocyte lipids. The data show that formation of an apoprotein-lipid complex at the air-water interface has both electrostatic and hydrophobic components. This contradicts results from other laboratories studying erythrocyte membrane recombination by bulk methods.     

A comparison of the effects of ionic strength on three preparations of acetylcholinesterase in the presence and absence of gallamine

The kinetic behaviour of three forms of acetylcholinesterase as a function of ionic strength of the medium was investigated. The forms of enzyme were that bound to human erythrocyte membranes, acetylcholinesterase solubilized from these by Triton X-100, and a commercial preparation of the enzyme from bovine erythrocytes. The properties investigated were hydrolysis of the substrate acetylthiocholine, decarbamylation of dimethylcarbamyl-acetylcholinesterase and ageing of isopropylmethylphosphonyl-acetylcholinesterase. The effect of 10^?5 M gallamine triethiodide on these properties was also examined as a function of ionic strength.Detailed results for the variation of kinetic behaviour with ionic strength and the presence of gallamine are presented. No unified theory to predict the influence of these variables on all three forms of the enzyme could be formulated. Thus, the enzyme conformation stabilized by gallamine at low ionic strength was not necessarily similar to that of the gallamine-free enzyme at physiological ionic strength. Nor was it useful to consider the free enzyme at low ionic strength to be a model of the membrane-bound enzyme in vivo (Crone, 1973).It was concluded that kinetic results for solubilized and partially or wholly purified acetylcholinesterase cannot be extrapolated to the membrane-bound enzyme. Prediction of the effect of drugs on the system in vivo requires the use of the membrane-bound enzyme.     

Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane.

Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD.     

Species-dependent differences in the effect of ionic strength on potassium transport of erythrocytes: the role of lipid composition.

The Rb+(K+) efflux of erythrocytes from six mammalian species was investigated in solutions of physiological and low ionic strength. A species dependent increase of the Rb+(K+) efflux in low ionic strength solution could be observed. The rate constant of Rb+(K+) efflux of erythrocytes in physiological ionic strength solution correlates with the content of arachidonic acid of the membrane phospholipids. The same relation was observed in solution of low ionic strength with the exception of human erythrocytes. In addition, an age-dependent correlation of the rate constant of Rb+(K+) efflux from calf erythrocytes in low ionic strength solution with the content of arachidonic acid of the membrane phospholipids was found. The Rb+(K+) efflux of human erythrocytes, which is enhanced in low ionic strength solution, decreases with the decreasing temperature. The temperature-dependent ESR order parameter of a fatty acid spin label for human and cow erythrocytes in solution of physiological and low ionic strength media suggested that the effect of low ionic strength on Rb+(K+) efflux is not solely based on a change of membrane fluidity. The results are interpreted as being due to a specific influence of membrane phospholipids on the Rb+(K+) efflux.     

Effect of Co2+-substitution on the substrate specificity of phospholipase C from Bacillus cereus during attack on two membrane systems.

Phospholipid degradation by native phospholipase C from Bacillus cereus and enzyme forms where one or both of the Zn2+ prosthetic groups had been replaced with Co2+ was studied in human erythrocyte membranes (ghosts) and resuspended freeze-dried bovine brain myelin. The rate of total phospholipid degradation was 2-9-fold more rapid with erythrocytes than with myelin. With both membrane systems the activity decreased in the order ZnZn-enzyme greater than ZnCo-enzyme greater than CoCo-enzyme. For all three enzyme forms with either membrane system, phosphatidylethanolamine (or the ethanolamine-containing phosphoglycerides) and phosphatidylcholine were hydrolysed most rapidly and sphingomyelin least. The relative rate of sphingomyelin degradation was highest with the ZnCo-enzyme. In myelin at low ionic strength there seemed to be a core of phospholipid that was very resistant to degradation by native phospholipase C but which was much more accessible to the Co2+-substituted forms. It is suggested that ZnCo-phospholipase C has potential applications in membrane studies.     

Application of high performance liquid chromatography to the analysis of cytoplasmic and nuclear oestrogen receptors

Soluble and nuclear preparations of fresh human endometrium have been analysed by HPLC on a gel permeation column. With high ionic strength eluants recoveries of receptor binding were less than 40% while low ionic strength eluants gave recoveries of 70% or greater. Non-specific binding by endometrial and plasma proteins was largely lost at both high and low ionic strength. Soluble binding components saturable by diethylstilboestrol (DES) were resolved into two components differing in heat lability and response to variations in ionic strength. Molybdate appears to alter the receptor protein rather than the activity of inactivating enzymes. At low ionic strength nuclear binding components saturable by DES could be resolved into two components.     

An improved purification procedure for calpastatin, the inhibitor protein specific for the intracellular calcium-dependent proteinases, calpains

The specific inhibitor protein (calpastatin) for the calcium-dependent intracellular proteinases (calpains) is an important regulator of these enzymes. In this communication we describe a one day procedure for purifying 3 to 5 mg of calpastatin from a kilogram of bovine myocardium. This represents a substantial improvement over previously described methods, and should facilitate future studies of calpastatin structure and function. A key, novel step in the purification was dye-matrix chromatography on an Affi-Gel Blue column. Contrary to previous indications, calpastatin purified by the new method did not contain significant amounts of carbohydrate. However, the presence of covalently bound phosphate in purified bovine myocardial calpastatin was confirmed and co-migration of phosphate and calpastatin activity was demonstrated on Bio-Gel A-1.5m chromatography. Thus, it is possible that calpastatin function is regulated by phosphorylation.     

Effects of preparation method on reversible conformational changes induced by neutral salts in spin-labeled erythrocyte membranes

Addition of “chaotropic” neutral salts to spin-labeled erythrocyte membranes produced changes in membrane conformation proportional to the known activities of the salts: KSCN > CaCl₂ ≥ KNO₃ ? NaCl. The effects had a threshold of about 0.10 m and increased through 0.60 m for all salts. Above 0.6 m other changes due to dielectric effects and/or protein loss began.The chaotropic effects were reversible on removal of the chaotropic ion, for human (or bovine) membranes prepared with a buffer ionic strength of 0.02. For membranes prepared with a buffer ionic strength of 0.005 the changes were irreversible. Exposure to salt concentration greater than 1 m also produced irreversible changes, probably due to solubilization. The results appear to show that the water-soluble membrane proteins are essential to maintain the “native” membrane conformation.     

The effect of cryogenic storage on human erythrocyte membrane proteins as determined by polyacrylamide-gel electrophoresis

A study was conducted to determine the effects of freezing on the major membrane proteins of isolated human erythrocyte membranes. Membranes in low or normal ionic strength medium were frozen at slow or fast freezing rates. The membrane protein composition and elution of proteins from the membranes were studied utilizing polyacrylamide-gel electrophoresis in a sodium dodecyl sulfate or an acetic acid-urea-phenol solvent system. Neither a change in the composition of the membrane proteins nor any elution of membrane protein during freezing and thawing was observed. The data indicate that any human erythrocyte membrane damage during freezing and thawing was not related to a change in major membrane protein composition. Human red cell membranes were stable at ?80 or ?196 °C in the absence of a cryoprotective agent.     

The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin)

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.