The interaction of human glycophorin with 8-anilino-1-naphthalene sulfonate

1977 ◽  
Vol 55 (9) ◽  
pp. 942-948 ◽  
Author(s):  
Jacob A. Verpoorte
Keyword(s):  
Sialic Acid ◽  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Erythrocyte Membranes ◽  
Ans Binding ◽  
Aromatic Residues ◽  
Apparent Binding Constant ◽  
Human Erythrocyte Membranes ◽  
Low Ionic Strength ◽  
Human Glycophorin

Both the sialoglycoprotein of human erythrocyte membranes, glycophorin, and the sialic acid free protein, obtained by treatment of glycophorin with neuraminidase (EC 3.2.1.18), increase the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). Binding of ANS to glycophorin is weak compared with the binding to bovine serum albumin (BSA). Equilibrium dialysis gives an apparent binding constant of about 4 × 103 M−1 at neutral pH, but Ka increases 1.75 times when NaCl or CaCl2 are added and 10-fold when the pH is lowered to 3.0. Sialic acid groups do not significantly affect ANS binding, although they have some effect at low ionic strength and neutral pH.Fluorescence studies indicate only one to two binding sites for ANS, with apparent pK = 3.8 ± 0.2. and located close to aromatic residues in glycophorin.Polarization and quantum efficiency of the fluorescence of ANS associated with glycophorin fail to indicate changes in the vicinity of the binding site when the pH is lowered.

The Membrane Binding of Morphine, Diphenylhydantoin, and Tetrahydrocannabinol

1972 ◽  
Vol 50 (12) ◽  
pp. 1193-1200 ◽  
Author(s):  
P. Seeman ◽  
M. Chau-Wong ◽  
S. Moyyen
Keyword(s):  
Ionic Strength ◽  
Partition Coefficient ◽  
Binding Sites ◽  
High Ionic Strength ◽  
Erythrocyte Membranes ◽  
Drug Concentrations ◽  
Human Erythrocyte Membranes ◽  
Low Ionic Strength ◽  
Frog Sciatic Nerve ◽  
Guinea Pig Brain

The adsorption of morphine to guinea pig brain synaptosome membranes and to human erythrocyte membranes was found to be passive, and unaffected by time, temperature, ouabain, dinitrophenol, saponin, or ATP. The membrane/buffer partition coefficient for morphine was 35 (at low ionic strength) and 1.2 (at high ionic strength). The synaptosome membrane/buffer partition coefficient for diphenylhydantoin was around 60 (at pH 8), while that for Δ9-tetrahydrocannabinol (THC) was around 380 (in the concentration range of around 10−5 M). The partition coefficient for the latter drug dropped by a factor of two or three with increasing drug concentrations for both erythrocyte ghosts and synaptosomes; there may be two types of binding sites for THC. The minimum blocking concentrations (frog sciatic nerve) were 1.1 × 10−2 M for morphine, and 8.3 × 10−4 M for diphenylhydantoin. The anesthetizing membrane concentrations of these two drugs are close to the value predicted by the Meyer–Overton rule for local anesthesia (30 mmol/kg dry membrane).

scholarly journals Ultrastructure of the intact skeleton of the human erythrocyte membrane.

1986 ◽  
Vol 102 (3) ◽  
pp. 997-1006 ◽  
Author(s):  
B W Shen ◽  
R Josephs ◽  
T L Steck
Keyword(s):  
Human Erythrocyte ◽  
Actin Filaments ◽  
Band 3 ◽  
Carbon Films ◽  
Erythrocyte Membranes ◽  
Accessory Proteins ◽  
Red Cell Membrane ◽  
Human Erythrocyte Membranes ◽  
Low Ionic Strength ◽  
Triton X

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

scholarly journals Membrane glycopeptides from old and young human erythrocytes (Short Communication)

1974 ◽  
Vol 140 (3) ◽  
pp. 557-560 ◽  
Author(s):  
Cesare Balduini ◽  
Carlo Luigi Balduini ◽  
Edoardo Ascari
Keyword(s):  
Sialic Acid ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Short Communication ◽  
Chemical Characterization ◽  
Erythrocyte Membranes ◽  
Main Peak ◽  
Human Erythrocyte Membranes ◽  
Papain Digestion

Glycopeptides were extracted by papain digestion from old and young human erythrocyte membranes and fractionated on DEAE-Sephadex A-25. Chemical characterization of the unfractionated samples and of the main peak eluted from the column indicates that glycoproteins of the erythrocyte membrane undergo significant decreases in sialic acid and galactosamine content with aging.

scholarly journals The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes

1982 ◽  
Vol 202 (1) ◽  
pp. 53-58 ◽  
Author(s):  
C. Peter Downes ◽  
Robert H. Michell
Keyword(s):  
Ionic Strength ◽  
Erythrocyte Membrane ◽  
Erythrocyte Membranes ◽  
Phosphodiesterase 4 ◽  
Maximal Activation ◽  
Human Erythrocyte Membranes ◽  
The Right ◽  
Phosphatidylinositol 4 ◽  
Low Ionic Strength ◽  
Micromolar Range

1. Both the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca2+ in the micromolar range. We have therefore compared the mechanisms and affinities for Ca2+ activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca2+-pump ATPase was activated by Ca2+ in a highly co-operative manner, with half-maximal activation occurring at about 0.3μm-Ca2+. At an optimal Ca2+concentration, calmodulin stimulated the Ca2+-sensitive ATPase activity about 10-fold. 3. Ca2+ activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca2+ requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca2+ activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg2+, half-maximal activation of the phosphodiesterase was at about 3μm-Ca2+. The presence of 1mm-Mg2+ shifted the Ca2+ activation curve to the right, as did elevation of the ionic strength. When the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg2+concentration, the ATPase was fully activated at 3μm-Ca2+, whereas no polyphosphoinositide phosphodiesterase activity was detected below 100μm-Ca2+. 5. The Ca2+-pump ATPase of the erythrocyte membrane normally maintains the Ca2+ concentration of healthy erythrocytes below approx. 0.1μm. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.

Effects of a series of alcohols on the binding of a fluorescent dye to erythrocyte membranes

1976 ◽  
Vol 54 (1) ◽  
pp. 35-41 ◽  
Author(s):  
S. H. Roth ◽  
L. Spero
Keyword(s):  
Membrane Proteins ◽  
Binding Sites ◽  
Erythrocyte Membranes ◽  
Membrane Action ◽  
Apparent Dissociation Constant ◽  
Ans Binding ◽  
Membrane Interaction ◽  
Low Concentrations ◽  
Anesthetic Concentration ◽  
Hypotonic Hemolysis

1. The effects of a series of aliphatic alcohols (methanol to octanol) on membrane proteins of erythrocytes were studied by monitoring the fluorescence of a dye (1-anilino-8-naphthalenesulfonic acid (ANS)) that adsorbs to erythrocyte ghost membranes. Low concentrations of all the alcohols reduced the ANS fluorescence of the membrane–ANS suspensions; lent to those which protect against hypotonic hemolysis on intact erythrocytes; higher concentrations markedly increased the fluorescence. Ethanol and methanol decreased ANS fluorescence at all concentrations.2. Lytic concentrations of saponin did not increase ANS fluorescence and did not modify the membrane action of the alcohols.3. None of these effects were observed in liposomes prepared from lipid extracts of the erythrocyte membrane.4. Since the apparent dissociation constant for the ANS–membrane interaction was unchanged in the presence of the alcohols, it was assumed that the fluorescence changes reflected alterations in the number of ANS binding sites. One conclusion is that low anesthetic concentration of the alcohols alter the conformation of membrane proteins, as indicated by the decreased number of ANS binding sites.

The determination of calcium-binding sites of human erythrocyte membranes

1984 ◽  
Vol 62 (6) ◽  
pp. 398-408 ◽  
Author(s):  
R. Blaine Moore ◽  
E. E. Dryden ◽  
D. I. C. Kells ◽  
J. F. Manery
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Plasma Membranes ◽  
Equilibrium Dialysis ◽  
Association Constants ◽  
Erythrocyte Membranes ◽  
Least Squares Regression ◽  
Calcium Binding Site ◽  
Calcium Binding Sites

Calcium binding to leaky erythrocyte plasma membranes was measured by three different procedures: Millipore filtration, equilibrium dialysis, and partition centrifugation. The curve derived from the binding equation, which best fit the means of the raw data, was used to estimate the association constants and capacities of the binding sites. A computer program (Gaushaus) which uses a nonlinear, least-squares regression protocol was also used to confirm these estimates. On the basis of these analyses we propose the presence of three classes of calcium-binding sites with the following apparent association constants and capacities: site 1, Ka = 3 × 104 M−1 and n = 30 nmol/mg protein; site 2, Ka = 3 × 103 M−1 and n = 200 nmol/mg protein; site 3, Ka = ~102 M−1 and n = ~200 nmol/mg protein. Calcium binding to erythrocyte membranes sealed in a high-salt solution showed the presence of site 3, but not site 2. The influence of phospholipids on the binding of calcium was evaluated by pretreating ghosts with phospholipase C (Clostridium welchii, EC 3.1.4.3). Treatment with this enzyme removed 80% of the total membrane phosphorus, predominantly from sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. By the method of partition centrifugation two classes of binding sites were identified by computer analysis. Their association constants and capacities are, respectively, 1.1 × 105 M−1 and 20 nmol/mg protein for site 1 and 4.4 × 103 M−1 and 200 nmol/mg protein for site 2. We speculate that calcium-binding site 1 is composed of acidic phospholipids, calcium-binding site 2 is composed of spectrin and actin, and calcium-binding site 3 is composed of sialic acid.

Structure of cytochalasins and cytochalasin B binding sites in human erythrocyte membranes

Biochemistry ◽  
1980 ◽  
Vol 19 (4) ◽  
pp. 679-683 ◽  
Author(s):  
Amrit L. Rampal ◽  
Harold B. Pinkofsky ◽  
Chan Y. Jung
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes

Studies on the Mechanism and Reversal of the Phospholipase-A2 Inactivation of D-Glucose Uptake by Isolated Human Erythrocyte Membranes

1974 ◽  
Vol 52 (12) ◽  
pp. 1097-1109 ◽  
Author(s):  
Batya Banjo ◽  
Caroline Walker ◽  
Ruth Rohrlick ◽  
Arthur Kahlenberg
Keyword(s):  
Fatty Acids ◽  
Glucose Uptake ◽  
Binding Sites ◽  
Phospholipase A ◽  
Enzyme Treatment ◽  
Erythrocyte Membranes ◽  
Membrane Phospholipids ◽  
Glucose Binding ◽  
Human Erythrocyte Membranes ◽  
By Products

The mechanism underlying the inactivation of the stereospecific uptake of D-glucose by isolated human-erythrocyte membranes following digestion with phospholipase A2 (Kahlenberg, A. &Banjo, B. (1972) J. Biol. Chem 247, 1156–1160) was investigated. This inactivation was not accompanied by any significant change in the uptake of L-glucose. The decrease in D-glucose uptake following limited (25–30%) cleavage of membrane phospholipids by phospholipase A2 was characterized by a twofold increase in the apparent dissociation constant of the D-glucose–membrane complex and a 34% decrease in the membrane's maximum capacity for D-glucose uptake. These effects of phospholipase A2 were completely reversed upon removal of the membrane-bound phospholipid byproducts (fatty acids and lysophospholipids) by washing the membranes with defatted bovine-serum albumin. Oleic acid and various lysophosphatides added to albumin-washed, phospholipase A2-treated membranes in amounts formed by the enzyme treatment produced negligible inhibition of D-glucose uptake. With more extensive phospholipase A2 digestion of membrane phospholipids, defatted bovine-serum albumin did not restore D-glucose uptake despite the removal of the phospholipid by-products formed.In addition to the inactivation of D-glucose uptake, limited enzyme treatment transforms the appearance of the membranes collected by centrifugation from opaque white to transparent and gelatinous. Both of these effects of phospholipase A2 are completely reversed upon incubation of the membranes at pH 5.5 for 2 h at 37 °C without loss of any of the membrane lysophosphatides and fatty acids formed by the enzyme treatment. It is suggested that this pH- and temperature-dependent restoration of D-glucose uptake is due to a conformational change resulting in the relocation of the membrane D-glucose-binding sites into a functional environment.These results indicate that the inactivation of D-glucose uptake by phospholipase A2, which was not accompanied by any change in L-glucose uptake, occurs by two different mechanisms. With limited hydrolysis of membrane phospholipids, one or both of the resulting phospholipid by-products reversibly inhibit the uptake of D-glucose by decreasing the affinity of the membrane for D-glucose and by masking a portion of the total available D-glucose-binding sites. However, upon extensive cleavage of phospholipids in the hydrophobic region of the membrane, there is an apparently irreversible disorganization of the membrane D-glucose-binding component. This might be due to destruction of vital phospholipids and/or a disturbance of the interactions between the lipid and protein components of the membrane.

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