Protein-disulfide isomerase- and protein thiol-dependent dehydroascorbate reduction and ascorbate accumulation in the lumen of the endoplasmic reticulum

The transport and intraluminal reduction of dehydroascorbate was investigat ed in microsomal vesicles from various tissues. The highest rates of transp ort and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These micro somes contain the highest amount of protein-disulfide isomerase, which is k nown to have a dehydroascorbate reductase activity, The steady-state level of intraluminal isotope accumulation was more than a-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-disul fide isomerase was similar, but the availability of protein thiols was diff erent and correlated with dehydroascorbate uptake. The increased isotope ac cumulation was accompanied by a higher rate of dehydroascorbate reduction a nd increased protein thiol oxidation in microsomes from diabetic animals, T he results suggest that both the activity of protein-disulfide isomerase an d the availability of protein thiols as reducing equivalents can play a cru cial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum, These findings also support the fact that dehydroascorbate can a ct as an oxidant in the protein-disulfide isomerase-catalyzed protein disul fide formation.