Abstract
The transport and intraluminal reduction of dehydroascorbate was investigated in microsomal vesicles from various tissues. The highest rates of transport and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These microsomes contain the highest amount of protein-disulfide isomerase, which is known to have a dehydroascorbate reductase activity. The steady-state level of intraluminal isotope accumulation was more than 2-fold higher in hepatic microsomes prepared from spontaneously diabetic BioBreeding/Worcester rats and was very low in fetal hepatic microsomes although the initial rate of transport was not changed. In these microsomes, the amount of protein-disulfide isomerase was similar, but the availability of protein thiols was different and correlated with dehydroascorbate uptake. The increased isotope accumulation was accompanied by a higher rate of dehydroascorbate reduction and increased protein thiol oxidation in microsomes from diabetic animals. The results suggest that both the activity of protein-disulfide isomerase and the availability of protein thiols as reducing equivalents can play a crucial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum. These findings also support the fact that dehydroascorbate can act as an oxidant in the protein-disulfide isomerase-catalyzed protein disulfide formation.
Highlights
The lumen of the endoplasmic reticulum (ER)1 and of the vesicular structures of the whole secretory pathway is characterized by an oxidizing environment reflected in a high ratio of glutathione disulfide versus glutathione [1, 2]
The highest DHA uptake was observed in adult rat liver microsomes, whereas in vesicles from other sources the uptake slightly exceeded or not exceed at all the level of the passive equilibrium (Table I)
The time course of the uptake was different; in nonhepatic microsomes the uptake reached a steady-state level in the first minute of incubation, whereas in adult rat liver microsomes a continuous accumulation could be observed (Fig. 1.) Ascorbate uptake was similar in all kinds of the investigated microsomal vesicles (Table I)
Summary
The lumen of the endoplasmic reticulum (ER) and of the vesicular structures of the whole secretory pathway is characterized by an oxidizing environment reflected in a high ratio of glutathione disulfide versus glutathione [1, 2]. Oxidizing conditions can be generated by the import of an oxidizing agent It is apparently inconsistent with the above facts that these compartments contain ascorbate, a reducing compound, at high concentrations [3,4,5]. The intraluminal accumulation of ascorbate can theoretically be explained by an active transport process or by its local generation from a membrane-permeable precursor. The aim of the present work was to explore the role of PDI in the intraluminal ascorbate accumulation in the ER. To this end, DHA transport, DHA reduction, and ascorbate accumulation were investigated in microsomes from various organs/cells having different PDI activities
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