Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter.
GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.
Katagiri
H
H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Asano
T
T
Shibasaki
Y
Y
Lin
J L
JL
Tsukuda
K
K
Ishihara
H
H
Akanuma
Y
Y
Takaku
F
F
Oka
Y
Y
D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western; D006224:Cricetinae; D003412:Cricetulus; D003571:Cytochalasin B / Q000378:metabolism*; D003847:Deoxyglucose / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D007930:Leucine / Q000235:genetics*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011817:Rabbits; D014364:Tryptophan / Q000235:genetics*
D000345:Affinity Labels; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D014364:Tryptophan; D003847:Deoxyglucose; D007930:Leucine