scholarly journals Hereditary spherocytosis of man. Altered binding of cytoskeletal components to the erythrocyte membrane

1982 ◽  
Vol 201 (2) ◽  
pp. 259-266 ◽  
Author(s):  
John S. Hill ◽  
William H. Sawyer ◽  
Geoffrey J. Howlett ◽  
James S. Wiley
Keyword(s):  
Ionic Strength ◽  
Membrane Protein ◽  
Hereditary Spherocytosis ◽  
Binding Capacity ◽  
Normal Subjects ◽  
Erythrocyte Membranes ◽  
Scatchard Analysis ◽  
Abnormal Cells ◽  
The Difference ◽  
Low Ionic Strength

Human erythrocytes possess a lattice work of extrinsic proteins on the inner face of the membrane (‘cytoskeleton’) that maintains the shape and deformability of the cell. The major proteins of the cytoskeleton are spectrin and actin, which are attached to the membrane by protein bands 2.1 (‘ankyrin’) and 4.1. The interactions of spectrin/actin with erythrocyte membranes from normal subjects and from patients with hereditary spherocytosis (HS) have been studied by using an air-driven ultracentrifuge, which can rapidly separate membranes from soluble proteins (150000g for 30s). The total amount of spectrin/actin in HS and normal ghosts is similar. However, the rate of dissociation of spectrin and actin from HS erythrocyte membranes at low ionic strength is significantly lower than that observed for normal membranes. Spectrin and actin isolated from either HS or normal membranes re-associated in a similar manner to spectrin/actin-depleted vesicles prepared from normal cells. Scatchard analysis showed an average binding capacity of 278μg/mg of membrane protein. However, spectrin/actin-depleted vesicles prepared from HS cells bound significantly less spectrin/actin prepared from either the normal or abnormal cells (average binding capacity 158μg/mg of membrane protein). The defect was defined further by studying the cytoskeleton obtained by Triton X-100 extraction of membranes. Under conditions of low ionic strength cytoskeletons prepared from HS membranes dissociated more slowly than those prepared from normal membranes, and only 80% of the protein from HS cytoskeletons could be solubilized after 180min compared with 100% for normal cytoskeletons. The difference between HS and normal membranes, which persists in isolated cytoskeletons, suggests that alterations in either the primary structure or the degree of phosphorylation of protein bands 2.1 or 4.1 may be central to the molecular basis of hereditary spherocytosis.

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.

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

INHIBITORY EFFECTS OF ITP SERA ON BINDING OF ANTIPLATELET GLYCOPROTEIN (GP) IIb/IIIa MONOCLONAL ANTIBODIES TO HUMAN UMBILICAL VASCULAR ENDOTHELIAL CELLS (HUVE)

1987 ◽  
Author(s):  
T Nakajima ◽  
T Koyama ◽  
Y Nishida ◽  
H Tanaka ◽  
E Kakishita ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vascular Endothelial Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Cell Pellet ◽  
A Cell ◽  
Molecular Masses ◽  
The Difference

Some ITP patients have specific autoantibodies to platelet GP IIb/IIIa. On the other hand, HUVE were shown to synthesize platelet GP IIb/IIIa like substances. Therefore, we studied the binding of ITP sera to HUVE by showing the inhibitory effect of ITP sera on the binding of anti-platelet GP IIb/IIIa monoclonal antibodies to HUVE. HUVE were cultured according to the method of Jaffe et al. 125-I-anti-platelet GP IIb/IIIa monoclonal antibody (125-I-Anti-GP) (40.3 mCi/mg), 40 yl, was added to a cell suspension of HUVE (1.5 × 104/500 μl) in a plastic RIA tube. After incubation for 30 min. at 4°C and centrifugation of 10,000 xg for 3 min., the radioactivity of the cell pellet was measured. Specific binding was determined by determining the difference between cell-bound radioactivity in the absence and presence of an excess amount of unlabelled ligand at 100 x concentrations. Scatchard analysis using 125-I-Anti-GP showed that the maximum binding capacity was 8 × 104/cell and Kd was 40.2 nM. The binding rate of 125-I-Anti-GP to HUVE treated with ITP (high PAIgG) sera (n=6) was 15.2±3.3% compared with 24.0±7.5%, observed for HUVE treated with normal sera (n=10). Treatment of ITP sera to HUVE significantly lowered the binding of 125-I-Anti-GP to HUVE (P<0.05). A combined analysis of SDS-PAGE and Western blotting of washed platelet and endothelial cell lysates shows that two proteins from each cells had similar or identical molecular masses to GP IIb/IIIa.These findings show that there are GP IIb/IIIa on the HUVE, ITP sera from our patients may have antibodies to HUVE GP IIb/IIIa and that anti-platelet GP IIb/IIIa antibodies in the ITP sera may bound not only to some platelets, but also to the HUVE

scholarly journals Phosphorylation in erythrocyte membranes from abnormally shaped cells

Blood ◽  
1976 ◽  
Vol 48 (6) ◽  
pp. 877-886 ◽  
Author(s):  
AC Greenquist ◽  
SB Shohet
Keyword(s):  
Membrane Protein ◽  
Protein Phosphorylation ◽  
No Reduction ◽  
Hereditary Spherocytosis ◽  
Erythrocyte Membranes ◽  
Hereditary Elliptocytosis

Erythrocyte protein phosphorylation was examined in membrane preparations of 25 patients with hereditary spherocytosis (HS). Reduced phosphorylation in substrate polypeptides was observed in 22 HS erythrocyte membranes for both splenectomized and nonsplenectomized patients. A reduction in labeling was also observed in a polypeptide which was labeled only in the presence of cAMP. No reduction was observed in membranes from immunologically acquired spherocytosis cells or from cells from patients with hereditary elliptocytosis. Heating membranes to 45 degrees C caused negligible inhibition of membrane protein phosphorylation, while heating to 50 degrees C extensively inhibited phosphorylation. Dephosphorylation of membrane protein that occurred in isolated membranes was not dependent upon cAMP.

scholarly journals The Study of Viability of Vibrio cholerae Strains in Low Ionic Strength Aquatic Environment

2019 ◽  
Author(s):  
Subha Sankar Paul ◽  
Eizo Takahashi ◽  
Goutam Chowdhury ◽  
Shin-ichi Miyoshi ◽  
Asish K. Mukhopadhyay ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Vibrio Cholerae ◽  
Aquatic Environment ◽  
Detection Rate ◽  
Environmental Water ◽  
Toxin Gene ◽  
Aquatic Medium ◽  
Vibrio Cholerae O1 ◽  
The Difference ◽  
Low Ionic Strength

It has been regarded that Vibrio cholerae O1 inhabit in environmental water. As many cholera patients emerge in Kolkata, it has been thought that V. cholerae O1 is easily detected in environmental water in Kolkata. However, the detection of V. cholerae O1 is rare, though other V. cholerae (NAG Vibrio) is constantly detected. To clear the reason for the difference of the detection rate of two Vibrios, we examined the viability of V. cholera O1 and NAG Vibrios in low ionic strength aquatic medium. We observed greater declining viability of V. cholerae O1 possessing cholera toxin gene (ctx) in low ionic strength solution, but the decline of NAG Vibrios non-possessing ctx is small. To evaluate the concerning of ctx in the viability, we examined the viabilities of V. cholerae O1which do not possess ctx and NAG Vibrios possessing ctx under the same condition. The result indicated that the existence of the ctx induces the decrease the viability of the host in low ionic strength solution. The decrease observed in this experiment might relate with the low detection of V. cholerae O1 possessing ctx in environmental water, though NAG Vibrio is constantly detected.

scholarly journals Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

1980 ◽  
Vol 86 (2) ◽  
pp. 694-698 ◽  
Author(s):  
C M Cohen ◽  
S F Foley
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Actin Filaments ◽  
Binding Capacity ◽  
Protein Band ◽  
Actin Binding ◽  
Erythrocyte Membranes ◽  
Cytoplasmic Surface ◽  
Independent Association ◽  
Inside Out

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain &lt; 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

scholarly journals Phosphorylation in erythrocyte membranes from abnormally shaped cells

Blood ◽  
1976 ◽  
Vol 48 (6) ◽  
pp. 877-886 ◽  
Author(s):  
AC Greenquist ◽  
SB Shohet
Keyword(s):  
Membrane Protein ◽  
Protein Phosphorylation ◽  
No Reduction ◽  
Hereditary Spherocytosis ◽  
Erythrocyte Membranes ◽  
Hereditary Elliptocytosis

Abstract Erythrocyte protein phosphorylation was examined in membrane preparations of 25 patients with hereditary spherocytosis (HS). Reduced phosphorylation in substrate polypeptides was observed in 22 HS erythrocyte membranes for both splenectomized and nonsplenectomized patients. A reduction in labeling was also observed in a polypeptide which was labeled only in the presence of cAMP. No reduction was observed in membranes from immunologically acquired spherocytosis cells or from cells from patients with hereditary elliptocytosis. Heating membranes to 45 degrees C caused negligible inhibition of membrane protein phosphorylation, while heating to 50 degrees C extensively inhibited phosphorylation. Dephosphorylation of membrane protein that occurred in isolated membranes was not dependent upon cAMP.

INHERITED DECREASE OF THE BINDING CAPACITY OF THYROXINE-BINDING GLOBULIN (TBG)

1970 ◽  
Vol 64 (1) ◽  
pp. 171-180 ◽  
Author(s):  
O. P. Heinonen ◽  
B.-A. Lamberg ◽  
J. Virtamo
Keyword(s):  
Autosomal Dominant ◽  
Binding Capacity ◽  
Normal Subjects ◽  
Free Thyroxine ◽  
Gradual Change ◽  
Normal Range ◽  
Thyroxine Binding Globulin ◽  
The Difference ◽  
Factor Governing ◽  
Mode Of Inheritance

ABSTRACT A family with a decrease in the binding capacity of thyroxine binding globulin (TBG) is described. The gene was probably transmitted by a female who married twice. Five subjects were considered TBG deficient, with TBG values ranging from 8.4 to 16.8 μg/100 ml. Of these subjects 2 were males and 3 females; the males had the lowest binding capacities. In addition, 1 male and 3 females had TBG values within the low normal range and were considered as possibly affected. The mode of inheritance could not be exactly defined but there were indications that it might be an autosomal dominant. The correlation of TBG to the protein-bound iodine in the serum (PBI), to the triiodothyronine uptake by Sephadex (T3-U), to the ratio between them (T3-U/PBI), and to the proportionate free thyroxine (PFT4) was strongly positive or negative. A gradual change in these variables from the affected to the unaffected subjects was observed. These correlations indicated that in normal subjects TBG is the most important factor governing the PBI and free thyroxine levels. In addition, a strong inverse and statistically significant correlation was observed between the binding capacity of TBG and that of the pre-albumin (TPBA). The difference between affected and unaffected subjects with regard to TPBA was also statistically significant.

scholarly journals The equilibrium constant of the phosphoglyceromutase reaction

1974 ◽  
Vol 139 (3) ◽  
pp. 491-497 ◽  
Author(s):  
John B. Clarke ◽  
Michael Birch ◽  
Hubert G. Britton
Keyword(s):  
Ionic Strength ◽  
Equilibrium Constant ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Binding Constants ◽  
Acid Dissociation ◽  
Effect Of Temperature ◽  
Acid Dissociation Constants ◽  
The Difference ◽  
Low Ionic Strength

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4–7.9), in solutions of different ionic strength (0.06–0.3) and in the presence of Mg2+, at 30°C and at 20°C. The values obtained (8.65–11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg2+-binding constants were measured spectrophotometrically and the values, 286mm-1 and 255mm-1 for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg2+ concentration and ionic strength there will be little variation in equilibrium constant.

Selective inhibition of [3H]nitrendipine binding to brain and cardiac membranes by bacitracin

1989 ◽  
Vol 67 (12) ◽  
pp. 1591-1595 ◽  
Author(s):  
Janet D. Smith ◽  
Gordon T. Bolger
Keyword(s):  
Ionic Strength ◽  
Rat Brain ◽  
Binding Sites ◽  
Selective Inhibition ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Brain Membranes ◽  
Cardiac Membranes ◽  
Low Ionic Strength

The effects of bacitracin were investigated on [3H]nitrendipine binding to rat brain and cardiac membranes in a low ionic strength (5 mM Tris–HCl) buffer. Bacitracin inhibited [3H]nitrendipine binding to rat brain and cardiac membranes with IC50 values of 400 ± 100 and 4600 ± 400 μg/mL, respectively. Scatchard analysis in brain membranes revealed that bacitracin inhibited [3H]nitrendipine binding primarily by reducing the Bmax but also by producing a small increase in the Kd. In brain membranes, Na+ (100 mM) and Ca2+ (2 mM) reduced the potency of bacitracin to inhibit [3H]nitrendipine binding by approximately sixfold with IC50 values of 2600 ± 300 and 2100 ± 400 μg/mL observed for bacitracin in the presence of 100 mM Na+ and 2 mM Ca2+, respectively. The EC50 values for the effects of Na+ and Ca2+ were 800 ± 200 μM and 25 ± 5 mM. K+, Mg2+, choline, and increasing the assay buffer of Tris–HCl to 50 mM also decreased the inhibition of [3H]nitrendipine binding by bacitracin. These results suggest that bacitracin specifically modulates [3H]nitrendipine binding in a cation-dependent manner and that brain and cardiac dihydropyridine binding sites are either biochemically different or exist in a different membrane environment.Key words: bacitracin, [3H]nitrendipine, brain, heart.

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