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

Sulfhydryl substituents of the human erythrocyte hexose transport mechanism

1986 ◽  
Vol 250 (6) ◽  
pp. C853-C860 ◽  
Author(s):  
R. E. Abbott ◽  
D. Schachter ◽  
E. R. Batt ◽  
M. Flamm
Keyword(s):  
Human Erythrocyte ◽  
Transport Mechanism ◽  
Cytochalasin B ◽  
Erythrocyte Membranes ◽  
Type I ◽  
Type Ii ◽  
Hexose Transport ◽  
Intact Cells ◽  
Type Iii ◽  
Human Erythrocyte Membranes

Sulfhydryl substituents of the hexose transport mechanism of human erythrocyte membranes were studied with membrane-impermeant and -permeant maleimide derivatives. Three sulfhydryl classes have been identified on the basis of their reactivity toward the reagents and their effects on the transport mechanism. Type I sulfhydryl is located at the outer (exofacial) surface of the membrane and bound covalently on treatment of intact cells with the membrane-impermeant glutathione-maleimide. This sulfhydryl is required for the transport, and it is protected from alkylation, i.e., its reactivity toward maleimides is decreased by the presence of D-glucose or cytochalasin B. Type II sulfhydryl is also required for the transport, but it differs from type I in that D-glucose (but not cytochalasin B) increases the reactivity toward maleimides. Further, it is located at the endofacial surface of the membrane, since reaction with glutathione-maleimide occurs only in leaky ghosts and not in intact cells. Alkylation by glutathione-maleimide of type I and type II sulfhydryls increases the half-saturation for the binding of D-glucose to erythrocyte membranes. In contrast, inactivation of type III sulfhydryls by N-ethylmaleimide or dipyridyl disulfide decreases the half-saturation concentration for the binding of D-glucose and other transported hexoses to the membranes; nontransported sugars are not affected similarly. Type III sulfhydryl is not inactivated by the polar reagent glutathione-maleimide and is probably located in a nonpolar domain of the transport mechanism. Inactivation of either type I or II sulfhydryls decreases or eliminates the flux asymmetry of the hexose transport mechanism.

scholarly journals Presence of peripheral-type benzodiazepine binding sites on human erythrocyte membranes

1988 ◽  
Vol 152 (1-2) ◽  
pp. 47-53 ◽  
Author(s):  
James M.M. Olson ◽  
Brian J. Ciliax ◽  
William R. Mancini ◽  
Anne B. Young
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Erythrocyte Membranes ◽  
Benzodiazepine Binding ◽  
Human Erythrocyte Membranes ◽  
Peripheral Type ◽  
Benzodiazepine Binding Sites

scholarly journals Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

1982 ◽  
Vol 93 (2) ◽  
pp. 463-469 ◽  
Author(s):  
P P da Silva ◽  
M R Torrisi
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Transmembrane Protein ◽  
Freeze Fracture ◽  
Membrane Skeleton ◽  
Erythrocyte Membranes ◽  
Con A ◽  
Human Erythrocyte Membranes

Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin-associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces. Label is sparse over the protoplasmic faces. These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face. Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freeze-fracture. This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton.

scholarly journals Erythrocyte nucleoside and sugar transport Endo-β-galactosidase and endoglycosidase-F digestion of partially purified human and pig transporter proteins

1986 ◽  
Vol 240 (2) ◽  
pp. 349-356 ◽  
Author(s):  
F Y Kwong ◽  
S A Baldwin ◽  
P R Scudder ◽  
S M Jarvis ◽  
M Y Choy ◽  
...  
Keyword(s):  
Human Erythrocyte ◽  
Cytochalasin B ◽  
Sugar Transport ◽  
Glucose Transporters ◽  
Nucleoside Transporters ◽  
Enzyme Treatment ◽  
Erythrocyte Membranes ◽  
Nucleoside Transporter ◽  
Human Erythrocyte Membranes ◽  
Beta Galactosidase

Nucleoside- and glucose-transport proteins isolated from human erythrocyte membranes were photoaffinity-labelled with [3H]nitrobenzylthioinosine and [3H]cytochalasin B, respectively, and subjected to endo-beta-galactosidase or endoglycosidase-F digestion. Without enzyme treatment the two radiolabelled transporters migrated on SDS/polyacrylamide gels with the same apparent Mr (average) of 55,000. Apparent Mr (average) values after endo-beta-galactosidase digestion were 47,000 and 48,000 for the nucleoside and glucose transporters respectively, and 44,000 and 45,000 respectively after endoglycosidase-F digestion. In contrast, endo-beta-galactosidase had no effect on the electrophoretic mobility of the nucleoside transporter isolated from pig erythrocytes. This transport system exhibited a higher Mr than the human protein, endoglycosidase-F treatment decreasing its apparent Mr (average) from 64,000 to 57,000. It is concluded that the human and pig erythrocyte nucleoside transporters are glycoproteins containing N-linked oligosaccharide. The data provide evidence of substantial carbohydrate and polypeptide differences between the human and pig erythrocyte nucleoside transporters, but evidence of molecular similarities between the human erythrocyte nucleoside and glucose transporters.

scholarly journals Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes

1987 ◽  
Vol 248 (1) ◽  
pp. 21-26 ◽  
Author(s):  
H Sato ◽  
S Aono ◽  
R Semba ◽  
S Kashiwamata
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Membrane Surface ◽  
Membrane Vesicles ◽  
Erythrocyte Membranes ◽  
Binding Properties ◽  
Before And After ◽  
Human Erythrocyte Membranes ◽  
Bilirubin Binding ◽  
The Right

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.

Characterization and solubilization of the specific binding sites for d-α-tocopherol from human erythrocyte membranes

1982 ◽  
Vol 31 (21) ◽  
pp. 3455-3461 ◽  
Author(s):  
Jayantha Wimalasena ◽  
Michael Davis ◽  
Abbas E. Kitabchi
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Specific Binding ◽  
Erythrocyte Membranes ◽  
Specific Binding Sites ◽  
Human Erythrocyte Membranes

scholarly journals Cytochalasin inhibits the rate of elongation of actin filament fragments

1979 ◽  
Vol 83 (3) ◽  
pp. 657-662 ◽  
Author(s):  
SS Brown ◽  
JA Spudich
Keyword(s):  
Actin Filament ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Cytochalasin D ◽  
Erythrocyte Membranes ◽  
Maximal Inhibition ◽  
Control Value ◽  
Human Erythrocyte Membranes ◽  
Final Extent ◽  
Assembly Rate

Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.

scholarly journals Monoclonal antibodies possibly recognize conformational changes in the human erythrocyte glucose transporter

1992 ◽  
Vol 281 (1) ◽  
pp. 103-106 ◽  
Author(s):  
H Nishimura ◽  
H Kuzuya ◽  
A Kosaki ◽  
M Okamoto ◽  
M Okamoto ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Conformational Changes ◽  
Human Erythrocyte ◽  
Binding Sites ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Tertiary Structure ◽  
Erythrocyte Membranes ◽  
C Terminus ◽  
Low Concentrations

Two monoclonal antibodies (MAG17 and MAG20) were raised against the human erythrocyte glucose transporter, which was purified on an immunoaffinity column using a polyclonal antibody to the C-terminal peptide (residues 477-492) of the glucose transporter of HepG2 cells. To obtain antibodies which recognize the native glucose transporter integrated in the membrane, hybridomas were screened both by e.l.i.s.a. with purified glucose transporter and by dot-blotting with erythrocyte membranes. The antibodies immunoprecipitated D-glucose-inhibitable [3H]cytochalasin B-photoaffinity-labelled glucose transporters, but did not recognize the transporter on Western blotting. The presence of the C-terminal peptide did not inhibit the binding of these antibodies to the glucose transporter, suggesting that the antibodies recognized sites different from the transporter C-terminus. D-Glucose (0.1-100 microM) inhibited the binding of MAG17 and MAG20 to the transporter by 50%, indicating that the conformation of the epitopes was altered allosterically by D-glucose. Cytochalasin B inhibited the binding of MAG17 to the transporter, but enhanced the binding of MAG20 at low concentrations (less than 0.02 microM). These data suggest that the glucose transporter has high- and low-affinity binding sites for D-glucose and cytochalasin B, and that binding of D-glucose and cytochalasin B induces conformational changes in the transporter. Monoclonal antibodies which recognize the tertiary structure of the glucose transporter can be used for investigating its function and structure when integrated in the membrane.

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