The parameters describing the transport kinetics for both 2-NBDG and d-glucose (dissociation constants, Michaelis–Menten constants, maximal rates of uptake and outflow) were calculated from the measurements made with 2-NBDG. 2-NBDG transport was asymmetric, consistent with GLUT1 being the major functional GLUT isoform transporting 2-NBDG in muscle fibres at rest. The rate constants were markedly reduced in the presence of 20 mM d-glucose to 0.0082 ± 0.0004 s -1 and 0.0056 ± 0.0002 s -1 in fast-twitch and slow-twitch fibres respectively. The time course of 2-NBDG uptake in the presence of 50 µM 2-NBDG follows a one-phase exponential plateau curve and is faster in fast-twitch (rate constant 0.053 ± 0.0024 s -1) than in slow-twitch fibres (rate constant 0.031 ± 0.0021 s -1). Fibres were identified as fast-twitch or slow-twitch based on the differential sensitivity of their contractile apparatus to Sr 2+. The 2-NBDG uptake was >90% suppressed by 100 µM cytochalasin B in both fast-twitch and slow-twitch fibres, indicating that the 2-NBDG transport is GLUT-mediated.
#John posterino in nj free
MethodsĪ novel method is described for measuring free 2-NBDG transport across plasma membrane of single rat muscle fibres at rest. To develop a method for direct measurement of the fluorescent d-glucose analogue 2-NBDG transport across the plasma membrane of single skeletal muscle fibres and derive the theoretical framework for determining the kinetic parameters for d-glucose transport under basal conditions.
0 kommentar(er)
