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 The human erythrocyte nucleoside and glucose transporters are both band 4.5 membrane polypeptides

1983 ◽  
Vol 214 (3) ◽  
pp. 995-997 ◽  
Author(s):  
J S R Wu ◽  
S M Jarvis ◽  
J D Young
Keyword(s):  
Human Erythrocyte ◽  
Glucose Transporters ◽  
Erythrocyte Membranes ◽  
Nucleoside Transporter ◽  
Hexose Transport ◽  
Major Site ◽  
Equilibrium Binding ◽  
Human Erythrocyte Membranes

Human erythrocyte membranes and partially purified nucleoside transporter (band 4.5 and 7) were photoaffinity labelled with 3H-labelled 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine under equilibrium binding conditions. Band 4.5 was the major site of radiolabelling in both preparations. These experiments provide additional evidence to implicate band 4.5 polypeptides in nucleoside permeation, proteins previously shown to be involved in hexose transport.

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.

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

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.

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