Jc. Vera et al., RESOLUTION OF THE FACILITATED TRANSPORT OF DEHYDROASCORBIC ACID FROM ITS INTRACELLULAR ACCUMULATION AS ASCORBIC-ACID, The Journal of biological chemistry, 270(40), 1995, pp. 23706-23712
We performed a detailed kinetic analysis of the uptake of dehydroascor
bic acid by HL-60 cells under experimental conditions that enabled the
differentiation of dehydroascorbic acid transport from the intracellu
lar reduction/accumulation of ascorbic acid. Immunoblotting and immuno
localization experiments identified GLUT1 as the main glucose transpor
ter expressed in the HL-60 cells. Kinetic analysis allowed the identif
ication of a single functional activity involved in the transport of d
ehydroascorbic acid in the HL-60 cells. Transport was inhibited in a c
ompetitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose.
In turn, dehydroascorbic acid competitively inhibited the transport of
both sugars. A second functional component identified in experiments
measuring the accumulation of ascorbic acid appears to be associated w
ith the intracellular reduction of dehydroascorbic acid to ascorbic ac
id and is not directly involved in the transport of dehydroascorbic ac
id via GLUT1. Transport of dehydroascorbic acid by HL-60 ells was inde
pendent of the presence of external Na+, whereas the intracellular acc
umulation of ascorbic acid was found to be a Na+-sensitive process. Th
us, the transport of dehydroascorbic acid via glucose transporters is
a Na+-independent process which is kinetically and biologically separa
ble from the reduction of dehydroascorbic acid to ascorbic acid and it
s subsequent intracellular accumulation.