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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 proteindisulfide isomerase-catalyzed protein disulfide formation.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 suitable redox properties of these organelles are necessary for the formation and the maintenance of disulfide bonds in the secretory and plasma membrane proteins. 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 (3-5). The intraluminal accumulation of ascorbate can theoretically be explained by an active transport process or by its local generation from a membrane-permeable precursor. Whereas there are no data supporting the first possibility in the ER, the facilitated diffusion of dehydroascorbate (DHA), the oxidized form of ascorbate, has been described in rat liver microsomes. DHA uptake is presumably mediated by the glucose transporter T3 subunit of the glucose-6-phosphatase system (6). Local ascorbate oxidation and DHA formation have also been observed in microsomal vesicles (7). Therefore, ascorbate accumulation can be attributed to the intraluminal reduction of DHA taken up. However, enzyme(s) participating in the process and the source(s) of the reducing equivalents are unknown. Protein-disulfide isomerase (PDI), a major protein of the ER lumen, is known to have DHA reductase activity (8, 9). The aim of the present work was to explore the role of PDI in the intraluminal ascorbate accumulation in the ...

Ascorbate synthesis causes glutathione consumption in the liver. Addition of gulonolactone resulted in an increase of ascorbate production in isolated murine hepatocytes. At the same time, a decrease in reduced glutathione (GSH) level was observed. In hepatic microsomal membranes, ascorbate synthesis stimulated by gulonolactone caused an almost equimolar consumption of GSH. This effect could be counteracted by the addition of catalase or mercaptosuccinate, indicating the role of hydrogen peroxide formed during ascorbate synthesis in the depletion of GSH. The observed phenomenon may be one of the reasons why the evolutionary loss of ascorbate synthesis could be advantageous.Key words: Ascorbic acid; Glutathione; Hydrogen peroxide; Gulonolactone oxidase; Evolution; Mouse liver amino acids (1 mM of each) under constant bubbling of gas (02/ CO2, 95 : 5, v/v) at 37°C. Incubations were terminated by the addition of TCA (5% final concentration). Microsomal experimentsHepatic microsomal membranes were prepared and stored according to [6]. Microsomes (usually 1 mg protein/ml) were incubated in 50 mM potassium phosphate buffer pH 7.4 containing 5 mM MgC12 at 37°C for 30 min. Incubations were terminated by the addition of TCA (5% final concentration). Metabolite measurementsAscorbate and GSH were measured in the trichloroacetic acid soluble supernatant of the cells or microsomes by the method of Omaye [7,8] and Ball [9], respectively. Microsomal oxygen consumption was detected polarographically at 37°C using a Clarke-type oxygen electrode. Microsomal hydrogen peroxide production was measured enzimatically according to [10] in the incubation medium mentioned above supplemented with 40 ~tg/ml horseradish peroxidase, 66 ~tg/ml o-dianisidine and 0.2 mM sodium azide.

The relationship between glutathione deficiency, glycogen metabolism and ascorbate synthesis was investigated in isolated murine hepatocytes. Glutathione deficiency caused by various agents increased ascorbate synthesis with a stimulation of glycogen breakdown. Increased ascorbate synthesis from UDP-glucose or gulonolactone could not be further affected by glutathione depletion. Fructose prevented the stimulated glycogenolysis and ascorbate synthesis caused by glutathione consumption. Reduction of oxidised glutathione by dithiothreitol decreased the elevated glycogenolysis and ascorbate synthesis in diamide or menadione treated hepatocytes. Our results suggest that a change in GSI-I/GSSG ratio seems to be a sufficient precondition of altering glycogenolysis and a consequent ascorbate synthesis.

The orientation of gulonolactone oxidase activity was investigated in rat liver microsomes. Ascorbate formation upon gulonolactone addition resulted in higher intravesicular than extravesicular ascorbate concentrations in native microsomal vesicles. The intraluminal ascorbate accumulation could be prevented or the accumulated ascorbate could be released by permeabilising the vesicles with the pore-forming alamethicin. The formation of the other product of the enzyme, hydrogen peroxide caused the preferential oxidation of intraluminal glutathione in glutathione-loaded microsomes. In conclusion, these results suggest that the orientation of the active site of gulonolactone oxidase is intraluminal and/or the enzyme releases its products towards the lumen of the endoplasmic reticulum.z 1998 Federation of European Biochemical Societies.

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Ascorbic acid synthesis was stimulated by glucagon, dibutyryl cyclic AMP, as well as phenylephrine, vasopressin or okadaic acid, in hepatocytes prepared from fed mice. However, no such effect was observed in glycogen-depleted cells from starved animals, either in the presence or absence of glucose. The rate of ascorbate synthesis showed close correlation with the glucose release by hepatocytes. In mice the injection of glucagon increased plasma ascorbate concentration fifteenfold, and caused a sixfold elevation of the ascorbate content of the liver. These results show that hepatic ascorbate synthesis is dependent on glycogenolysis, and indicate a regulatory role of ascorbate released by the liver.

The transport of glucuronides synthesized in the luminal compartment of the endoplasmic reticulum by UDP-glucuronosyltransferase isoenzymes was studied in rat liver microsomal vesicles. Microsomal vesicles were loaded with p-nitrophenol glucuronide (5 mM), phenolphthalein glucuronide or UDP-glucuronic acid, by a freeze-thawing method. In was shown that: (i) the loading procedure resulted in millimolar intravesicular concentrations of the different loading compounds; (ii) addition of UDP-glucuronic acid (5 mM) to the vesicles released both intravesicular glucuronides within 1 min; (iii) glucuronides stimulated the release of UDP-glucuronic acid from UDP acid-loaded microsomal vesicles; (iv) trans-stimulation of UDP-glucuronic acid entry by loading of microsomal vesicles with p-nitrophenol glucuronide, phenolphthalein glucuronide, UDP-glucuronic acid and UDP-N-acetyl-glucosamine almost completely abolished the latency of UDP-glucuronosyltransferase, although mannose 6-phosphatase latency remained unaltered; (v) the loading compounds by themselves did not stimulate UDP-glucuronosyltransferase activity. This study indicates that glucuronides synthesized in the lumen of endoplasmic reticulum can leave by an antiport, which concurrently transports USP-glucuronic acid into the lumen of the endoplasmic reticulum.