TABLE 2.

Ligand binding to GLUT1

Experimenta	Cytochalasin B bindingb		d-Glucose bindingcKi(app)
	Kd(app)	Bmax
	nm	nm	mm
Control	133 ± 18	68 ± 9	8.6 ± 1.2
Post-trypsin	495 ± 80	118 ± 15	3.1 ± 0.5
Post-trypsin/CB	474 ± 73	119 ± 11	5.0 ± 0.8
α-Chymotrypsin	—	—	—

^a[³H]Cytochalasin B binding to GLUT1 proteoliposomes. CB binding was measured using GLUT1 proteoliposomes in saline buffer (control); GLUT1 proteoliposomes were incubated with trypsin to remove fragments L1, L2, L6, N and C termini, and TM1 (trypsin); GLUT1 was trypsinized in the presence of 10 μm CB to additionally remove TM8 (post-trypsin/CB); and GLUT1 proteoliposomes were treated with α-chymotrypsin. Results are shown as mean ± S.E. “—” indicates that CB binding was not detected

^bCB binding was measured in triplicate at 50–5000 nm CB. Two or more separate experiments were made per condition, and the entire data set was averaged for each [CB] in each condition. Binding displays simple Michaelis-Menten kinetics. Binding isotherms were analyzed by nonlinear regression to obtain the maximum binding and half-saturation constants (B_max and K_d(app), respectively)

^cBinding was also measured in the presence of d-glucose, a competitive inhibitor of CB binding to GLUT1 (19). K_i(app) for d-glucose (Glc) inhibition of CB binding was calculated from R, the ratio (CB bound in the absence of Glc):(CB bound when Glc is present), as K_i(app) = [Glc]/(((R(K_d(app) + [CB])) – [CB])/K_d(app) – 1)