GLUT1 transmembrane glucose pathway. Affinity labeling with a transportable D-glucose diazirine.
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 Km of 3.2 mM and the Vmax of 3.2 micromol/s/ml. D-Glucose inhibited the 3-DAG flux competitively with an apparent KI of 11 mM. Cytochalasin B inhibited this 3-DAG flux in a dose-dependent manner with an estimated KI of 2.4 x 10(-7) M. 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 mM D-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.
Lachaal
M
M
Biophysics Laboratory, Veterans Administration Medical Center, Buffalo, New York 14215, USA.
Rampal
A L
AL
Lee
W
W
Shi
Y
Y
Jung
C Y
CY
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D001391:Azo Compounds; D001665:Binding Sites; D001692:Biological Transport; D001786:Blood Glucose / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005947:Glucose / Q000031:analogs & derivatives; D051272:Glucose Transporter Type 1; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification
C098668:3-deoxy-3,3-aziglucopyranose; D000345:Affinity Labels; D001391:Azo Compounds; D001786:Blood Glucose; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; C495677:SLC2A1 protein, human; D005947:Glucose