Analysis of ATP modulation of lysine modification. (A) Time course of GLUT1 labeling by sulfo-NHS-LC-biotin. GLUT1 proteoliposomes were preincubated in buffer lacking (○) or containing 4 mM AMP (•) or 4 mM ATP (▪) before addition of sulfo-NHS-LC-biotin. Ordinate, extent of labeling (measured as OD415); abscissa, time in minutes. Results are shown as mean ± SEM of at least four determinations. Biotinylation kinetics follow pseudo-first order kinetics where labeling equals B0 + B∞(1 − e−kt). The curves have the following constants: buffer (○), B0 = 0.15 ± 0.02, B∞ = 1.09 ± 0.05, k = 0.0023 ± 0.003 per minute; AMP (•), B0 = 0.16 ± 0.01, B∞ = 1.20 ± 0.03, k = 0.0019 ± 0.0001 per minute; ATP (▪), B0 = 0.098 ± 0.009, B∞ = 0.66 ± 0.02, k = 0.0022 ± 0.0002 per minute. (A, inset) Average B∞ (±SEM) of at least three labeling experiments made in triplicate for control, 4 mM AMP, and 4 mM ATP conditions. (B) Effects of nucleotides on equilibrium GLUT1 biotinylation. Ordinate, extent of total biotin incorporation (shown as mean ± SEM; n = 3 or greater); abscissa, [AMP] or [ATP] (mM) present during labeling. The pseudo-first-order rate constant describing GLUT1 labeling by sulfo-NHS-LC-biotin is unaffected by nucleotides. The extent of labeling is not significantly affected by AMP alone (•). Assuming labeling is described by BC − BN[nucleotide]/(Ki + [nucleotide]), nonlinear regression analysis indicates that for labeling in the presence of ATP (▪), BC = 1.210 ± 0.007, BN = 0.72 ± 0.04, and Ki = 2.1 ± 0.1 mM. ATP inhibition of labeling was also measured in the presence of 2 mM AMP (▵), where BC = 0.9, BN = 0.6, and Ki = 3.8 ± 1.5 mM. AMP therefore anatagonizes ATP modulation of biotinylation with Ki(app) for AMP = 2.2 mM.