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(Received for publication, July 23, 1996, and in revised form, February 12, 1997)
From the Human myeloid leukemia cells (HL-60) transport
only the oxidized form of vitamin C (dehydroascorbic acid) and
accumulate the vitamin in the reduced form, ascorbic acid. We performed
a detailed study of the role of glutathione in the intracellular
trapping/accumulation of ascorbic acid in HL-60 cells. Uptake studies
using HL-60 cells depleted of glutathione by treatment with
L-buthionine-(S,R)
sulfoximine and diethyl maleate, revealed no changes in the cells'
ability to transport dehydroascorbic acid and accumulate ascorbic acid. Similar transport and accumulation rates were obtained using HL-60 cells containing intracellular glutathione concentrations from 6 mM to 1 µM. HL-60 cells, containing as little
as 5 µM glutathione, were able to accumulate up to 150 mM ascorbic acid intracellularly when incubated with
dehydroascorbic acid. Glutathione was capable of reducing
dehydroascorbic acid by a direct chemical reaction, but only when
present in a greater than 10-fold stoichiometric excess over
dehydroascorbic acid. The accumulation of ascorbic acid by HL-60 cells
was strongly temperature-dependent and was very inefficient
at 16 °C. On the other hand, the direct chemical reduction of
dehydroascorbic acid by excess glutathione proceeded efficiently at
temperatures of 16 °C. Our data indicate that
glutathione-dependent reductases in HL-60 cells are not
responsible for the ability of these cells to accumulate millimolar
concentrations of ascorbic acid. These findings indicate that
alternative enzymatic mechanisms are involved in the cellular reduction
of dehydroascorbic acid.
Volume 272, Number 15,
Issue of April 11, 1997
pp. 9915-9921
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,§
and
Program in Molecular Pharmacology and
Therapeutics and the § Department of Medicine, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
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