title: Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol.
abstract: Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.
authors.affiliation: Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Pérez
authors.forename: Paola
authors.initials: P
authors.lastname: Ojeda
authors.forename: Ximena
authors.initials: X
authors.lastname: Valenzuela
authors.forename: Marcela
authors.initials: M
authors.lastname: Ortega
authors.forename: Claudio
authors.initials: C
authors.lastname: Sánchez
authors.forename: Lorena
authors.initials: L
authors.lastname: Ojeda
authors.forename: Maite
authors.initials: M
authors.lastname: Castro
authors.forename: Juan G
authors.initials: JG
authors.lastname: Cárcamo
authors.forename: M Cecilia
authors.initials: MC
authors.lastname: Rauch
authors.forename: Ilona I
authors.initials: II
authors.lastname: Concha
authors.forename: Coralia I
authors.initials: CI
authors.lastname: Rivas
authors.forename: Juan C
authors.initials: JC
authors.lastname: Vera
authors.forename: Alejandro M
authors.initials: AM
authors.lastname: Reyes
mesh_terms: D019325:3-O-Methylglucose / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003571:Cytochalasin B / Q000378:metabolism; D003847:Deoxyglucose / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006072:Gossypol / Q000378:metabolism / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009536:Niacinamide / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection
chemical_list: D015703:Antigens, CD; D051272:Glucose Transporter Type 1; C495677:SLC2A1 protein, human; D019325:3-O-Methylglucose; D009536:Niacinamide; D003571:Cytochalasin B; D003847:Deoxyglucose; C435941:INSR protein, human; D011972:Receptor, Insulin; D005947:Glucose; D006072:Gossypol