Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.
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 temperaturedependent, 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 GLUTtype transporter. The intravesicular reduction of dehydroascorbate leads to the accumulation of ascorbate and contributes to the low intraluminal reduced/oxidized glutathione ratio.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 (1-3). 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. Such transporters have been thoroughly investigated in plasma membrane of different cells (4 -9) and in chromaffin granula (10), but the transport of ascorbate and dehydroascorbate in microsomes has not been described in detail. 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. EXPERIMENTAL PROCEDURESPreparation of Rat Liver Microsomes-Microsomes were prepared from 24-h fasted male Sprague-Dawley rats (180 -230 g) as reported (11). Microsomal fractions were resuspended in buffer A (100 mM KCl, 20 mM NaCl, 1 mM MgCl 2 , 20 mM MOPS, 1 pH 7.2). The suspensions (60 -80 mg of protein/ml) were rapidly frozen and maintained under liquid N 2 until used. Intactness of microsomal vesicles was checked by measuring the latency of mannose-6-phosphatase (12) and p-nitrophenol UDP-glucuronosyltransferase activity (13), they were greater than 95% in all the p...
Beyond its general role as antioxidant, specific functions of ascorbate are compartmentalized within the eukaryotic cell. The list of organelle-specific functions of ascorbate has been recently expanded with the epigenetic role exerted as a cofactor for DNA and histone demethylases in the nucleus. Compartmentation necessitates the transport through intracellular membranes; members of the GLUT family and sodium-vitamin C cotransporters mediate the permeation of dehydroascorbic acid and ascorbate, respectively. Recent observations show that increased consumption and/or hindered entrance of ascorbate in/to a compartment results in pathological alterations partially resembling to scurvy, thus diseases of ascorbate compartmentation can exist. The review focuses on the reactions and transporters that can modulate ascorbate concentration and redox state in three compartments: endoplasmic reticulum, mitochondria and nucleus. By introducing the relevant experimental and clinical findings we make an attempt to coin the term of ascorbate compartmentation disease.
Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose-6-phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.z 1998 Federation of European Biochemical Societies.
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