Rat Erythrocyte Membrane Research Articles

O-83) - β-adrenergic receptors, adenylate cyclase activity and guanine nucleotide regulatory proteins in rat reticulocyte and erythrocyte membranes

O-83) - β-adrenergic receptors, adenylate cyclase activity and guanine nucleotide regulatory proteins in rat reticulocyte and erythrocyte membranes

Calcium-induced proteolysis of spectrin and band 3 protein in rat erythrocyte membranes

Calcium-dependent protease activity capable of degrading a number of endogenous proteins was found in rat red blood cell membranes. This protease activity, like that found in human red blood cells, was activated by low concentrations of calcium, but in the rat red blood cells, unlike the human red blood cells, calcium-activated protease activity was membrane-bound. A number of endogenous membrane-bound proteins were degraded after the addition of calcium to the membranes. These included spectrin bands 1 and 2 as well as bands 3, 2.1, and 2.2. No calcium-induced aggregation (transglutaminase activity) was noted in the rat red blood cell membranes.

Chloral hydrate inhibition in vitro of ATPase in membrane of rat erythrocytes and in microsomes of dog kidney external medulla

The study of the general anesthetic chloral hydrate and its effects on rat erythrocyte membranes and dog kidney microsomes showed that ATPases were reversibly inhibited in every case. The inhibition was cooperative in the cases of (Mg 2− + Na − + K +)-ATPase, Mg 2−-ATPase and (Na + + K −)-ATPase of rat erythrocyte membrane, while Ca 2−-ATPase and (Mg 2− + Ca 2−)-ATPase were non-cooperative. The chloral hydrate concentrations necessary to diminish the activity of the enzyme to half of the V max (I 50) were 6 mM for Ca 2+-ATPase from erythrocyte membranes and 82 mM for Mg 2−-ATPase from intact external kidney medulla microsomes. When Ca 2−-ATPase was studied in the absence of Mg 2+ in these microsomes, the affinity for Ca 2− was very low, but the enzyme was sensitive to chloral hydrate.

Lignocaine: Inhibitory effect on synaptosomal and erythrocyte membrane-bound acetylcholinesterase activity

Lignocaine (5–20 mM) reversibly inhibits ( in vitro) AChE activity of rat brain synaptosome (33–66%) and erythrocyte membrane (10–54%) in a concentration dependent manner. Lineweaver-Burk plots indicate that lignocaine-induced inhibition of AChE activity in synaptosome is competitive, whereas in erythrocyte membrane inhibition of AChE is non-competitive in nature. Arrhenius plots show that transition temperatures of both synaptosomal and erythrocyte membrane-bound AChE are significantly reduced in the presence of lignocaine. These results suggest that lignocaine increases the lipid fluidity of synaptosomal and erythrocyte membrane which may be a cause of inhibition of membrane-bound AChE activity.

The effect of reducing agents on the structure of mammalian β-adrenergic receptors

Under reducing conditions (5% β-mercaptoethanol) the mammalian β-adrenergic receptor binding site from both β 1 (porcine heart membranes) and β 2 receptors (hamster lung and rat erythrocyte membranes) appears to reside on peptides of M r 62,000–65,000 as determined by photoaffinity labeling with p-azido-m-[ 125I]iodobenzylcarazolol and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When similar experiments are performed in these same systems under a variety of non-reducing conditions, there are minimal changes in the apparent molecular weight of both the β 1- and β 2-adrenergic receptor binding subunits and no specifically labeled higher molecular weight proteins are observed suggesting that there are no disulfide linked subunits in mammalian β-adrenergic receptors.

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT −) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT − erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT − erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT − erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.

High affinity L-triiodothyronine binding sites on washed rat erythrocyte membranes.

Two orders of saturable binding sites for L-triiodothyronine were found on washed rat erythrocyte membranes. The high affinity, low capacity site showed values of Kd 0.19 X 10(-10) M. This value was in the range of concentration of free L-triiodothyronine found in the plasma and was several orders of magnitude higher than the Kd values previously reported for other L-triiodothyronine membrane-binding systems. The binding site also showed a high stereospecificity for L-triiodothyronine. D-3,5,3'-triiodothyronine and L-thyroxine were less potent (about 1000-fold) than L-triiodothyronine in competing for these sites. L-3,3,5'-triiodothyronine and triiodothyroacetic acid were inactive. The physiological relevance of this site is considered.

Erythrocyte membrane in rats fed high erucic acid-containing mustard oil: Osmotic fragility, lipid composition, and (Na +, K +)- and (Ca 2+, Mg 2+)-ATPases

Erythrocyte hemolytic properties, cholesterol/phospholipid ratios, fatty acid composition, and activities of the membrane-bound enzymes (Na +, K +)- and (Ca 2+, Mg 2+)-ATPase were studied in male and female rats fed low erucic acid rapeseed (LEAR) and high erucic acid mustard oils (HEAM) for a period of 16 months. Rats receiving groudnut oil (GNO) served as controls. Erythrocytes from HEAM-receiving male and female rats showed increased resistance to hypotonic hemolysis. In male rats this was associated with an 85% increase ( P < 0.07) in the cholesterol/phospholipid molar ratio. The fatty acid double-bond index showed an increase in male rats receiving HEAM as well as LEAR oils. In the erythrocytes from female rats, the cholesterol/phospholipid molar ratio and double bond index remained unaffected. Specific activity of ouabain-sensitive (Na +, K +)-ATPase showed a small (+20%) but significant ( P < 0.05) increase in male but not female rats of HEAM group. Total (Na +, K +)-ATPase, ouabain-insensitive component, and (Ca 2+, Mg 2+)-ATPase were not altered in rats receiving LEAR or HEAM.

Properties of rat erythrocyte membrane cytoskeletal structures produced by digitonin extraction: digitonin-insoluble beta-adrenergic receptor, adenylate cyclase, and cholera toxin substrate.

Rat erythrocyte plasma membranes have been extracted exhaustively with digitonin at low temperature, and the residual, detergent-extracted membrane cytoskeletal material is compared to that prepared with Triton X-100 with respect to protein, glycoprotein, phospholipid, and cholesterol content. Digitonin, a weaker detergent than Triton X-100, solubilizes only 26% of the phospholipids and none of the cholesterol. SDS-polyacrylamide gel electrophoresis reveals that differences between the proteins extracted by the two detergents are primarily quantitative. In terms of functional preservation, digitonin retains in the cytoskeleton 28% of the beta-adrenergic receptor binding activity (with the balance accounted for in the supernatant), greater than 90% of the adenylate cyclase and greater than 90% of the 45,000 mol wt polypeptide cholera toxin substrate. The cytoskeletal-associated beat-adrenergic receptor retains binding properties for antagonist and agonist which are identical to those of the native membrane receptor. The digitonin-extracted cytoskeleton containing the beta-adrenergic receptor may provide a useful vehicle for the reconstitution of a hormone-sensitive adenylate cyclase.

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Structural studies of the acidic oligosaccharides

Among the four acidic oligosaccharide fractions obtained by paper electrophoresis of the hydrazinolysate of the plasma membrane glycoproteins of rat erythrocytes, one was further separated into two by prolonged paper electrophoresis using 120-cm paper. Three fractions were mixtures of monosialyl oligosaccharides and two of disialyl oligosaccharides. After desialylation, their neutral portions were fractionated by Bio-Gel P-4 column chromatography and by affinity chromatography using a Con A-Sepharose column. Structural studies of the neutral oligosaccharides, thus obtained, indicated that at least 26 different complex-type oligosaccharides are present as a neutral portion of the acid oligosaccharides. Structurally they can be classified into bi-, tri-, and tetraantennary oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc OT as their common cores. Galβ1 → 3Galβ1 → 4GlcNAc, Siaα2 → 3Galβ1 → 4GlcNAc, Siaα2 → 6Galβ1 → 4GlcNAc, and a series of Siaα2 → (Galβ1 → 4GlcNAcβ1 → 3) n · Galβ1 → 4GlcNAc were found as their outer chains. Their structures together with the structures of neutral oligosaccharides reported in the preceding paper indicated that the outer chain moieties of the asparagine-linked sugar chains of rat erythrocyte membrane glycoproteins are formed not by random concerted action of glycosyl transferases in Golgi membrane but by the mechanism in which the formation of one outer chain will regulate the elongation of others.

State of rat erythrocyte membrane Na, K-ATPase activity in botulinum toxin poisoning

State of rat erythrocyte membrane Na, K-ATPase activity in botulinum toxin poisoning

Effect of thyroid status on β-adrenergic receptor, adenylate cyclase activity and guanine nucleotide regulatory unit in rat cardiac and erythrocyte membranes

The effect of thyroid hormone on the β-receptor coupled adenylate cyclase in rat crude cardiac membranes was analysed by measuring the number of DHA-binding sites, adenylate cyclase activity and the amount of cholera toxin catalysed ADP-ribosylation of a protein with a molecular weight of 42,000 in cardiac and erythrocyte membranes. In crude rat cardiac membranes, the number of DHA-binding sites (78 ± 15 fmole/mg protein in the euthyroid state) is increased to 158 ± 20 fmole/mg protein in the hyperthyroid state and decreased to 51 ± 6 fmole/mg protein in the hypothyroid state; the affinity of the binding sites remained unchanged ( K D 2.9−4.3 nM). l-Isoprenaline (10 −4 M)-stimulated adenylate cyclase activity varied in parallel to the number of DHA-binding sites in hyper- and euthyroidism. Thyroid hormone, however, did not influence GppNHp (10 −4 M)-stimulated adenylate cyclase activity. Cholera toxin catalysed ADP-ribosylation of normal crude cardiac membranes resulted in a 1.8 fold increase in adenylate cyclase activity in the presence of GTP (10 −4) and l-isoprenaline (10 −4M), presumably as a result of inhibition of GTPase. In crude cardiac membranes cholera toxin catalyses the ADP-ribosylation of one major protein, which comigrates on sodium dodecylsulfate-polyacrylamide gel electrophoresis with the putative regulatory component of adenylate cyclase (mol. wt 42,000). In different thyroid states the amount of the regulatory component (as determined by cholera toxin dependent labelling) was equal (112 fmole/mg protein in euthyroid crude cardiac membranes). Basal activity of adenylate cyclase showed a significant difference between activity in euthyroid (3.7 ± 0.2 pmole cAMP/mg protein/min) and hypothyroid (5.4 ± 0.2 pmole cAMP/mg protein/min), but not in hyperthyroid crude cardiac membranes (3.4 ± 0.2 pmole cAMP/mg protein/min). Our results indicate, that thyroid hormone regulates the number of DHA-binding sites and basal activity (in hypothyroidism) in crude cardiac membranes and thereby causes different results in l-isoprenaline-induced adenylate cyclase activity.

Carbonic anhydrase activity in mitochondria from rat liver.

An 18O exchange method has been used to determine the location of carbonic anhydrase in mitochondria from rat liver and to examine the role of this enzyme in the kinetics of CO2 in resting and respiring mitochondria. Using digitonin subfractionation, we have determined that a substantial fraction, 40 to 60%, of the carbonic anhydrase activity in the mitochondrion from rat liver is located in the space between the inner and outer membranes; the remaining activity was found in the matrix with no detectable activity in the sedimented membranes. The total catalytic CO2 hydration activity measured in intact mitochondria from rat liver was about 1% of that found in an equal volume of rat erythrocytes. The apparent permeability constant representing the barrier for the diffusion of HCO3(-) from external solution to intramitochondrial carbonic anhydrase, 9 X 10(-5) cm s-1, is near in magnitude to the permeability constant for the diffusion of HCO3(-) across the rat erythrocyte membrane, 4 X 10(-4) cm s-2. Calcium-induced respiratory jumps were shown to cause changes in the rate of 18O exchange between CO2 and H2O that were consistent with a net uptake of CO2 by the mitochondria.

Mapping of mammalian β-adrenoreceptors by use of macromolecular alprenolol derivatives: A comparison with amphibian erythrocyte receptors

The interaction of macromolecular alprenolol derivatives with β-adrenoreeeptors of rat heart, lung, and erythrocytes and frog erythrocytes has been studied. Macromolecular derivatives were prepared by covalently coupling alprenolol to dextrans containing a homologous series of spacer arms of various lengths. The affinity of these macromolecules for frog erythrocyte membranes increased with increasing length of spacer arm. In contrast, the affinity of these macromolecules for all mammalian membrane preparations was weak and insensitive to the length of the spacer arm. The inhibition of [ 3H]dihydroalprenolol binding to rat heart, lung, and erythrocyte membrane preparations by these macromolecular derivatives was more than 1000-fold less potent than inhibition by alprenolol. The results suggest different structural characteristics between mammalian and amphibian β-adrenoreceptors; however, apparently only small differences between mammalian receptors could be distinguished with these probes.

Selective changes in fatty acid composition of phosphatidylserine in rat erythrocyte membrane induced by nitrate

The relationship between nitrate which is formed from inhaled nitrogen dioxide, a common air pollutant, and changes in fatty acid metabolism of phosphatidylserine in rat erythrocytes has been examined. When erythrocytes were incubated at 37°C for 60 min with fatty acid, the incorporation rate of [1- 14C]arachidonic acid and [9,10- 3H]palmitic acid into phosphatidylserine was 15% (80 pmol/h per μmol lipid phosphorus) and 20% (12 pmol/h per μmol lipid phosphorus) of those into phosphatidylethanolamine, respectively. By the addition of 1.0 mM sodium nitrate or 0.5 μM ionophore A23187 to the incubation mixture, the rate of incorporation of both arachidonic acid and palmitic acid into phosphatidylethanolamine was stimulated 1.45-fold. On the other hand, the incorporation of palmitic acid into phosphatidylserine was little affected, while that of arachidonic acid was stimulated 1.35-fold. An increase in arachidonic acid of phosphatidylserine was also found by the addition of nitrate or ionophore A23187. This increase was dependent on the concentration of extracellular calcium and observed by the addition of other chaotropic anions in the order SCN >CIO 4 − > NO 3 −. It seems likely, therefore, that nitrate causes changes in erythrocyte membranes to facilitate calcium uptake. Increasing the concentration of intracellular calcium may cause stimulation of acyl-CoA:lysophospholipid acyltransferase and/or endogenous phospholipase A 2.

Copper(II)-catalyzed lipid peroxidation in liposomes and erythrocyte membranes.

Cu++ was uniquely capable of catalyzing the peroxidation of rat erythrocyte membrane lipid in the presence of 10 mM H2O2, whereas several other transition metal ions were without significant effect. In contrast, peroxidation of soybean phospholipid liposomes could be catalyzed with decreasing efficiency by Co++, Cu++, Pb++, or Cr+++ also in the presence of H2O2. The effect of imidazole on Cu++- catalyzed lipid peroxidation was stimulatory in liposomes and inhibitory in membrane preparations, whereas EDTA, histidine, citrate and alanine inhibited peroxidation in both systems. EDTA could stop the peroxidation after initiation, but catalase could not, indicating that Cu++ alone was necessary for the propagation of the chain reaction. Competitive inhibition studies with various scavengers of hydroxyl radicals or singlet oxygen and the absence of significant reaction enhancement by D2O indicated that neither of these reactive oxygen species was a major mediator in the Cu++-H2O2 oxidative system. A copper-oxygen complex may be directly involved in the initiation of peroxidation. Normal erythrocyte membranes and phospholipid liposomes also differ in their sensitivities toward external oxidative stress. In the absence of H2O2, CU++ (0.2 mM) was capable of catalyzing lipid peroxidation in liposomes, aged erythrocyte membranes and membranes from vitamin-E-deficient rats; however, freshly prepared membranes from control rats and liposomes containing alpha-tocopherol required H2O2 greater than 2 mM for the catalytic effect of Cu++ to be observed.

A heterophile system in human renal transplantation. X. HTA sensitivity includes sensitivity to human B lymphocytes.

Antibodies that react with heterophile transplantation antigen (HTA) have been shown previously not to react with HLA-A, B, or C antigens. This paper presents evidence that anti-HTA does react with a subpopulation of human lymphocytes which is comprised primarily of B cells. Anti-HTA reactivity was removed from sera by absorption with each of three different human B lymphocyte cell lines, but it was unaffected by absorption with platelets or thymocytes. Selected high titer anti-HTA sera absorbed with human platelets, human blood group type AB erythrocytes, and sheep erythrocytes caused lysis of a lymphocyte subpopulation principally composed of B lymphocytes. Absorption of these sera with rat erythrocytes removed both lymphocytic activity and anti-HTA activity. Antibody recovered by affinity purification with rat erythrocyte membrane preparations contained both lymphocytic and anti-HTA reactivity. These data, considered with previous studies, seem to establish that B cell sensitization may be acquired by a substantial segment of the population by natural immunization from enteric flora and/or by infections with enteric bacteria.

Effects of diabetes and high fat-high cholesterol diet on plasma lipid levels and on erythrocyte membrane composition

Erythrocyte membrane composition was studied in rats subjected to experimental hyperlipidemia and/or hyperglycemia by means of 6 weeks of high fat (40% w w )-high cholesterol (5% w w ) diet with and without 8 weeks of streptozotocin-induced diabetes. High fat-high cholesterol diet lowered plasma glucose levels in control and in diabetic animals. While the atherogenic diet produced only hypercholesterolemia, the same diet fed to diabetic animals produced both hypercholesterolemia and hypertriglyceridemia. The membrane protein content was lower in diabetic rats than in controls, while the cholesterol and phospholipids were higher in diabetic rat erythrocyte membranes. Feeding the atherogenic diet increased membrane lipid levels in only nondiabetic animals. The total carbohydrate content of the membranes was greater in diabetic animals than controls. Difference in relative proportion of individual sugars, e.g., galactose, mannose, glucose, and fucose of the membranes were observed between diabetic and control groups. These observations suggest that rat erythrocyte membrane composition is altered both in hyperglycemic and hyperlipidemic conditions, and may provide a useful model for evaluating lipid-carbohydrate abnormalities of membrane structures in diabetes mellitus.

Metabolism of phosphoinositides in the rat erythrocyte membrane: A reappraisal of the effect of magnesium on the 32P incorporation into polyphosphoinositides

The metabolism of phosphoinositides was investigated in the red blood cell membrane of the rat by measuring 32 P-incorporation into phospholipids after incubation of membranes with [γ- 32P]ATP in a medium containing magnesium. A new chromatographic procedure has been developed which facilitates the separation of triphosphoinositide, diphosphoinositide and phosphatidylinositol from the phospholipids present in lipid extracts of incubated ‘ghost’ membranes. Under our experimental conditions only two phospholipids, diphosphoinositide and triphosphoinositide, were 32P-labelled. Furthermore, the results indicate that either di- or triphosphoinositide could be labelled preferentially, depending upon the magnesium concentration of the incubation medium. This clarifies some apparent discrepancies reported in the literature between the 32P labelling of polyphosphoinositides observed in intact erythrocytes and that observed with ‘ghost’ membranes. In addition, the enzymatic pathways involved in the phosphoinositide metabolism are discussed.

Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane.

Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane was studied. The transport properties, such as pH-dependence and effects of reagents reacting with amino-groups, were similar to those to anions like Cl- through the human erythrocyte membrane. The rate of transport of anions through rat erythrocyte membranes is higher than through those of other mammals, such as guinea pig and bovine erythrocyte membranes. This relatively high rate of transport makes the rat erythrocyte membrane suitable for use in comparative studies on the transport of slowly penetrating substances, such as organic anions. The transport velocities of benezenesulfonic acid derivatives were compared with their physico-chemical properties. It was shown that the hydrophobicity has no effect on the transport, but the electronic property has a significant effect: the transport rate is mainly dependent on the e- donor capacities. This feature is the inverse to the well-known inhibitory effect of these derivatives on other anion transport: the inhibition is mainly dependent on the e- acceptor capacities. It is suggested that the transport is regulated by the binding capacity of anions to the transport site.