We synthesized a transportable diazirine derivative of D-glucose, 3-deoxy-3,3-azi-D-glucopyranose (3-DAG), and studied its interaction with purified human erythrocyte facilitative glucose transporter, GLUT1. 3-DAG was rapidly transported into human erythrocytes and their resealed ghosts in the dark via a mercuric chloride-inhibitable mechanism and with a speed comparable with that of 3-O-methyl-D-glucose (3-OMG). The rate of 3-DAG transport in resealed ghosts was a saturable function of 3-DAG concentration with an apparent K of 3.2 mM and the V of 3.2 µmol/s/ml. D-Glucose inhibited the 3-DAG flux competitively with an apparent K of 11 mM. Cytochalasin B inhibited this 3-DAG flux in a dose-dependent manner with an estimated K of 2.4 10M. Cytochalasin E had no effect. These findings clearly establish that 3-DAG is a good substrate of GLUT1. UV irradiation of purified GLUT1 in liposomes in the presence of 3-DAG produced a significant covalent incorporation of 3-DAG into GLUT1, and 200 mMD-glucose abolished this 3-DAG incorporation. Analyses of trypsin and endoproteinase Lys-C digestion of 3-DAG-photolabeled GLUT1 revealed that the cleavage products corresponding to the residues 115-183, 256-300, and 301-451 of the GLUT1 sequence were labeled by 3-DAG, demonstrating that not only the C-terminal half but also the N-terminal half of the transmembrane domain participate in the putative substrate channel formation. 3-DAG may be useful in further identification of the amino acid residues that form the substrate channel of this and other members of the facilitative glucose transporter family.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				R. F. Epand, R. M. Epand, and C. Y. Jung Ligand-modulation of the stability of the glucose transporter GLUT 1 Protein Sci., July 1, 2001; 10(7): 1363 - 1369. [Abstract] [Full Text] [PDF]

				A. C. Ward, Y. M. van Aesch, A. M. Schelen, and I. P. Touw Defective Internalization and Sustained Activation of Truncated Granulocyte Colony-Stimulating Factor Receptor Found in Severe Congenital Neutropenia/Acute Myeloid Leukemia Blood, January 15, 1999; 93(2): 447 - 458. [Abstract] [Full Text] [PDF]

				A. Seyfang, M. P. Kavanaugh, and S. M. Landfear Aspartate 19 and Glutamate 121 Are Critical for Transport Function of the myo-Inositol/H+ Symporter from Leishmania donovani J. Biol. Chem., September 26, 1997; 272(39): 24210 - 24215. [Abstract] [Full Text] [PDF]