Abstract
Ascorbate and dehydroascorbate transport was investigated in rat liver microsomal vesicles using radiolabeled compounds and a rapid filtration method. The uptake of both compounds was time- and temperature-dependent, and saturable. Ascorbate uptake did not reach complete equilibrium, it had low affinity and high capacity. Ascorbate influx could not be inhibited by glucose, dehydroascorbate, or glucose transport inhibitors (phloretin, cytochalasin B) but it was reduced by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and by the alkylating agent N-ethylmaleimide. Ascorbate uptake could be stimulated by ferric iron and could be diminished by reducing agents (dithiothreitol, reduced glutathione). In contrast, dehydroascorbate uptake exceeded the level of passive equilibrium, it had high affinity and low capacity. Glucose cis inhibited and trans stimulated the uptake. Glucose transport inhibitors were also effective. The presence of intravesicular reducing compounds increased, while extravesicular reducing environment decreased dehydroascorbate influx. Our results suggest that dehydroascorbate transport is preferred in hepatic endoplasmic reticulum and it is mediated by a GLUT-type transporter. The intravesicular reduction of dehydroascorbate leads to the accumulation of ascorbate and contributes to the low intraluminal reduced/oxidized glutathione ratio.
Highlights
Ascorbate producing and utilizing pathways are connected to the endomembrane system of the cell
The final enzymatic steps of ascorbate synthesis are located in the endoplasmic reticulum of hepatocytes or kidney cells; enzymes utilizing ascorbate or its oxidized form dehydroascorbate are characteristic proteins of the lumen [1,2,3]
The time course of the uptake processes showed that dehydroascorbate uptake exceeded the level of the passive equilibrium (3.5 nmol/mg protein; calculated from the intravesicular water space of microsomal vesicles: 3.5 l/mg protein)
Summary
Ascorbate producing and utilizing pathways are connected to the endomembrane system of the cell. The final enzymatic steps of ascorbate synthesis are located in the endoplasmic reticulum of hepatocytes or kidney cells; enzymes utilizing ascorbate (prolyl-3-hydroxylase, prolyl-4-hydroxylase, and lysyl hydroxylase) or its oxidized form dehydroascorbate (protein disulfide isomerase) are characteristic proteins of the lumen [1,2,3]. Their presence in the lumen is necessary for the posttranslational modification and folding of many proteins. Since ascorbate and dehydroascorbate are charged water-soluble compounds, transporter(s) should exist for their permeation through biological membranes. The aim of the present study was to detect and characterize the activity of the possible ascorbate and/or dehydroascorbate transporter(s) in the endoplasmic reticulum
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