ABSTRACT: Glucose-6-phosphatase (G6Pase) is a multiple protein complex in the endoplasmic reticulum (ER) that includes a mechanism (known as T3) for glucose exit from the ER to the cytosol. The molecular identity of T3 is not known. T3 has been shown to be functional in the absence of GLUT2, indicating that it is not GLUT2. Here we found a 55-kDa protein in high-density microsomal fraction (HDM) of rat hepatocytes that is recognized by polyclonal GLUT2 antibody raised against the GLUT2 C-terminal 14-amino-acid-sequence peptide. HDM contained calnexin but no integrin-beta1 or Na/K ATPase in Western blotting. Significant GLUT2 immunoreactivity was colocalized with colligin, an ER marker, in confocal microscopy. Furthermore, the 55-kDa protein in HDM was labeled with a covalently reactive, impermeable glucose transporter substrate, 1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl 4-benzoyl-benzoate (B3GL) when hepatocyte homogenates, but not intact cells, were labeled. In addition glucose efflux from HDM vesicles was sensitive to B3GL treatment in a dose-dependent manner. Based on these findings, we suggest that T3 may be a novel facilitative glucose transporter that is highly homologous to GLUT2 in the C-terminal sequence, thus cross-reacting with the GLUT2 antibody. The finding will be useful in molecular identification and cloning of T3.
Biochimica et Biophysica Acta 09/2002; 1564(1):198-206. · 4.66 Impact Factor
ABSTRACT: 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.
Journal of Biological Chemistry 02/1996; 271(9):5225-5230. · 4.77 Impact Factor
ABSTRACT: Bacterial glucokinase (GK) binds to purified, human erythrocyte glucose transporter (GT) reconstituted in vesicles. The binding is largely abolished if GT is predigested with trypsin, indicating that GK binds to the cytoplasmic domain of GT. The binding is a saturable function of GK concentration showing two distinct affinities with apparent KD of 0.33 and 5.1 μM. The binding is stimulated by an increasing concentration of ADP with the 50% maximal effect at 5 mM. Glucose-6-phosphate (G6P) also stimulates the binding with a distinct optimum at 25 mM. The binding is stimulated only slightly by ATP. D-glucose has no affect on the binding. KCl enhances the binding with the maximal effect at physiological intracellular concentrations. The binding is sensitive to changes in pH with an optimum at pH 4. The binding causes no detectable functional change in GT. However, the enzymatic activity of GK measured at nanomolar concentrations of GK is significantly greater in the presence of GT vesicles than in its absence or in the presence of protein-free vesicles, indicating that GK interacts with GT at this low concentration range with an apparent KD of 10 mM. Although its physiological significance is not known, the GK-GT interaction in vitro described here suggests that these two proteins may also interact in the cell and regulate carbohydrate metabolism. © 1993 Wiley-Liss, Inc.
Journal of Cellular Physiology 07/1993; 156(2):326 - 332. · 3.87 Impact Factor