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.

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 Calcium binding by human erythrocyte membranes

1971 ◽  
Vol 124 (3) ◽  
pp. 563-571 ◽  
Author(s):  
J. Forstner ◽  
J. F. Manery
Keyword(s):  
Ionic Strength ◽  
Aqueous Phase ◽  
Human Erythrocyte ◽  
Calcium Binding ◽  
Incubation Medium ◽  
Phospholipid Fraction ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes ◽  
Chloroform Methanol

1. The characteristics of Ca2+ binding to haemoglobin-free human erythrocyte membranes were investigated by using 45Ca and centrifugation partition of ‘ghosts’ from their external incubation medium. Equilibrium of ‘ghosts’ with external Ca2+ required less than 15min. 2. The binding did not vary with temperature in the range 0–37°C. 3. At pH7.4 ‘ghosts’ bound a maximum of 283μmol of Ca2+/g of ‘ghost’ protein, equivalent to 6.85×107 Ca2+ ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca2+ binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca2+ bound. 6. Extraction of 45Ca-labelled ‘ghosts’ with chloroform–methanol showed that the distribution of Ca2+ was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca2+ (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca2+: phosphorus ratio of about 1:2.

scholarly journals Interaction of platelet plasma membranes with thrombin-activated platelets

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 305-312 ◽  
Author(s):  
HR Prasanna ◽  
HH Edwards ◽  
DR Phillips
Keyword(s):  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Erythrocyte Membranes ◽  
Functional Sites ◽  
Activated Platelets ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes

Abstract This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

Sign in / Sign up

Export Citation Format

Share Document