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.

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.

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.

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.

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.

The binding sites of cytochalasin D. II. Their relationship to hexose transport and to cytochalasin B

1977 ◽  
Vol 91 (2) ◽  
pp. 239-248 ◽  
Author(s):  
Janet Tannenbaum ◽  
Stuart W. Tanenbaum ◽  
Gabriel C. Godman
Keyword(s):  
Binding Sites ◽  
Cytochalasin B ◽  
Cytochalasin D ◽  
Hexose Transport
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