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.

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 Solubilization of Native Actin Monomers from Human Erythrocyte Membranes

1986 ◽  
Vol 39 (2) ◽  
pp. 117 ◽  
Author(s):  
Leann Tilley ◽  
Margaret Dwyer ◽  
GB Ralston
Keyword(s):  
Ionic Strength ◽  
Human Erythrocyte ◽  
Dnase I ◽  
Erythrocyte Membranes ◽  
Dnase 1 ◽  
Human Erythrocyte Membranes ◽  
Low Ionic Strength

Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0�4 mM ATP and 0�05 mM calcium. In the absence of calcium and A TP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37�C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse 1.

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.

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.

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 Allosteric control of Zymomonas mobilis glucose-6-phosphate dehydrogenase by phosphoenolpyruvate

1997 ◽  
Vol 326 (3) ◽  
pp. 731-735 ◽  
Author(s):  
Roberts K. SCOPES
Keyword(s):  
Ionic Strength ◽  
Zymomonas Mobilis ◽  
Phosphate Concentration ◽  
Hill Coefficient ◽  
Allosteric Control ◽  
Low Glucose ◽  
Low Ionic Strength ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
T State ◽  
Micromolar Range

The second enzyme of the Entner–Doudoroff glycolytic pathway in Zymomonas mobilis, glucose-6-phosphate dehydrogenase, has been found to be inhibited by phosphoenolpyruvate (PEP). In the presence of PEP levels in the micromolar range, the response of the enzyme to glucose 6-phosphate concentration becomes sigmoidal, with a Hill coefficient up to 2. At low ionic strength in the absence of PEP, the response to glucose 6-phosphate concentration is Michaelis–Menten, but at physiological ionic strength and pH, a Hill coefficient of 1.3 to 1.4 was found even in the absence of PEP. Km values for NAD+ and NADP+ are also ionic-strength-dependent, increasing rapidly as salt concentration increases. Some sigmoidicity was also observed for NAD+ in the presence of PEP at low glucose 6-phosphate concentrations. The results can be interpreted in a Monod–Wyman–Changeux model, in which glucose 6-phosphate binds principally to the R-state, PEP to the T-state, and NAD+ to both states. These observations are clearly physiologically significant, and provide an explanation for the control of the balance between glycolytic throughput and ATP consumption in Z. mobilis.

scholarly journals Electron microscope study of reassociation of spectrin and actin with the human erythrocyte membrane

1981 ◽  
Vol 90 (1) ◽  
pp. 70-77 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
S Sato ◽  
M Nakao
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membrane ◽  
Thin Sections ◽  
Fixation Treatment ◽  
Human Erythrocyte Membranes ◽  
Inside Out

Reassociation of spectrin and actin with human erythrocyte membranes was studied by stereoscopic electron microscopy of thin sections combined with tannic acid- glutaraldehyde fixation. Treatment of the erythrocyte membrane with 0.1 mM EDTA (pH 8.0) extracted more than 90 percent of the spectrin and actin and concomitantly removed filamentous meshworks underlying the membranes, followed by fragmentation into small inside-out vesicles. When such spectrin-depleted vesicles were incubated with the EDTA extract (crude spectrin), a filamentous meshwork, similar to those of the original membranes, was reformed on the cytoplasmic surface of the vesicles. The filamentous components, with a uniform thickness of 9 nm, took a tortuous course and joined one another often in an end-to-end fashion to form a irregular but continuous meshwork parallel to the membrane. Purified spectrin was also reassociated with the vesicles in a population density of filamentous components almost comparable to that of the crude spectrin-reassociated vesicles. However, the meshwork formation was much smaller in extent, showing many independent filamentous components closely applied to the vesicle surface. When muscle G-actin was added to the crude spectrin- or purified spectrin- reassociated vesicles under conditions which favor actin polymerization, actin filaments were seen to attach to the vesicles through the filamentous components. Two modes of association of actin filaments with the membrane were seen: end-to-membrane and side-to- membrane associations. In the end-to-membrane association, each actin filament was bound with several filamentous components exhibiting a spiderlike configuration, which was considered to be the unit of the filamentous meshwork of the original erythrocyte membrane.

scholarly journals Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

1982 ◽  
Vol 92 (3) ◽  
pp. 714-721 ◽  
Author(s):  
Y Lange ◽  
RA Hadesman ◽  
TL Steck
Keyword(s):  
Ionic Strength ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Time Course ◽  
Isotonic Saline ◽  
Human Erythrocyte Membrane ◽  
Wide Range ◽  
Cell Stability ◽  
Mechanically Stabilized ◽  
Low Ionic Strength

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.

Coisolation of glycophorin A and polyphosphoinositides from human erythrocyte membranes

1978 ◽  
Vol 56 (5) ◽  
pp. 349-351 ◽  
Author(s):  
J. Thomas Buckley
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Lipid Composition ◽  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Firm Conclusion ◽  
Membrane Phospholipids ◽  
Human Erythrocyte Membranes ◽  
Significant Enrichment

The lipid composition of purified erythrocyte membrane glycophorin was measured. Diphosphoinositide, triphosphoinositide, and phosphatidylserine are the major phospholipids in glycophorin preparations. Nearly all of the radioactive diphosphoinositide and triphosphoinositide extracted from erythrocyte membranes by lithium diiodosalicylate are recovered in purified glycophorin. There appeared to be no significant enrichment of other acidic membrane phospholipids in the protein. The results do not permit a firm conclusion as to whether the polyphosphoinositides are associated specifically with the membrane protein or whether fortuitous binding has occurred during purification.

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