Microsomal Transport Research Articles

Mutation analysis in glycogen storage disease type 1 non-a

We report molecular and clinical findings in 13 patients with rare types of glycogen storage disease 1 (GSD1 non-a). Analysis of G6PT encoding a microsomal transporter protein has revealed mutations on both chromosomes in each case, four of which are novel. Diagnosis has been confirmed in three patients suspected of having GSD1 non-a without enzymatic studies involving liver biopsy, thus emphasising the advantage of G6PT mutation analysis for all GSD1 non-a patients.

Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-proton pump that has not yet been characterized at the molecular level. We previously cloned a gene (Atp6i, for V-proton pump, H+ transporting (vacuolar proton pump) member I) encoding a putative osteoclast-specific proton pump subunit, termed OC-116kD (ref. 4). Here we show that targeted disruption of Atp6i in mice results in severe osteopetrosis. Atp6i-/- osteoclast-like cells (OCLs) lose the function of extracellular acidification, but retain intracellular lysosomal proton pump activity. The pH in Atp6i-/- liver lysosomes and proton transport in microsomes of Atp6i-/- kidney are identical to that in wild-type mice. Atp6i-/- mice exhibit a normal acid-base balance in blood and urine. Our results demonstrate that Atp6i is unique and necessary for osteoclast-mediated extracellular acidification.

Molecular diagnosis of type 1c glycogen storage disease.

Glycogen storage disease type 1 (GSD 1) results from deficiency of the microsomal multicomponent glucose-6-phosphatase system. Malfunction of the catalytic subunit characterises GSD 1a. GSD 1b and GSD 1c are characterised by defective microsomal glucose-6-phosphate or pyrophosphate/phosphate transport, respectively. Recently, a gene encoding a microsomal transporter protein has been found to be mutated in GSD 1b and 1c patients. Here, we report the genomic sequence of the transporter gene and the detection of a homozygous 2-bp deletion (1211delCT) and a homozygous donor splice site mutation (317+1G-->T) in two GSD 1c patients, confirming that GSD 1c is allelic to GSD 1b.

Molecular Analysis of Glycogen Storage Disease Type Ib: Identification of a Prevalent Mutation among Japanese Patients and Assignment of a Putative Glucose-6-Phosphate Translocase Gene to Chromosome 11

Glycogen storage disease type Ib (GSD-Ib) is an inborn error of metabolism with autosomal recessive inheritance, caused by defects in microsomal transport of glucose-6-phosphate. Recently, Gerin et al isolated a human cDNA encoding a putative transporter homologous to bacterial transporters of hexose-6-phosphate, and identified two mutations in its gene in two patients with GSD-Ib (9). Independently, a linkage analysis mapped the GSD-Ib gene on chromosome 11q23 (10). It remains to be elucidated whether the two genes are identical or GSD-Ib is genetically heterogeneous. We first mapped the transporter gene on chromosome 11 by using a DNA panel of human/hamster hybridoma cells. The result suggested that the GSD-Ib genes identified by the two distinct approaches may be identical and GSD-Ib was allelic. We then studied four unrelated Japanese families with GSD-Ib, and found three novel mutations: a four-base deletion/two-base insertion, a point mutation within a consensus splicing donor site, and a missense mutation (W118R). The W118R mutation was found in 4 out of 8 mutant alleles, suggesting that it is prevalent among Japanese patients.

Plant cell membranes as biochemical targets of the phytotoxin helminthosporol.

Helminthosporol is one of the natural sesquiterpenoid toxins isolated and identified in the culture medium of the phytopathogenic ascomycete fungus Cochliobolus sativus. The effect of this phytotoxin was investigated on enzymatic activities, electron and ion transport in mitochondria, chloroplasts, and microsomes of plant. The results indicate that helminthosporol drastically affects the membrane permeability of these organelles to protons and substrate anions, inhibiting the mitochondrial oxidative phosphorylation, the photophosphorylation in chloroplasts, and the proton pumping across the cell plasma membrane. The 1,3-beta-glucan synthase activity, involved in defense mechanisms of plant cells against stress and damage, e.g., during pathogen attack, was also strongly inhibited by the toxin.

Liver microsomal transport of glucose-6-phosphate, glucose, and phosphate in type 1 glycogen storage disease.

The transport of glucose-6-phosphate (G6P), glucose, and orthophosphate into liver microsomes, isolated from six patients with various subtypes of type 1 glycogen storage disease (GSD), was measured using a light-scattering method. We found that G6P, glucose, and phosphate could all cross the microsomal membrane, in four cases of type 1a GSD. In contrast, liver microsomal transport of G6P and phosphate was deficient in the GSD 1b and 1c patients, respectively. These results support the involvement of multiple proteins (and genes) in GSD type 1. The results obtained with the light-scattering method are in accordance with conventional kinetic analysis of the microsomal glucose-6-phosphatase system. Therefore, this technique could be used to directly diagnose type 1b and 1c GSD.

Liver Microsomal Transport of Glucose-6-Phosphate, Glucose, and Phosphate in Type 1 Glycogen Storage Diseases

Liver Microsomal Transport of Glucose-6-Phosphate, Glucose, and Phosphate in Type 1 Glycogen Storage Diseases

Novel peptide-binding proteins and peptide transport in normal and TAP-deficient microsomes.

Most major histocompatibility complex (MHC) class I-binding peptides are translocated by TAP heterodimers, but some enter the ER lumen by alternative pathways. To further define mechanisms of peptide handling, we developed a system for the analysis of peptide-binding components in the ER membrane and lumen using iodinated cross-linkable peptide derivatives. Here we demonstrate that at least three proteins bind peptides in the ER lumen. Peptide cross-linking to these lumenal proteins can be used as an alternative method to monitor peptide transport. TAP and one other protein bind peptides on the cytoplasmic face of the ER. The presence of multiple peptide-binding proteins necessitates caution in interpreting traditional peptide-binding and transport assays. Finally, we demonstrate sequence-specific peptide transport in TAP-deficient cells transfected with only rat TAP1.

Calcium transport in microsomes isolated from rat parotid gland. Effects of ADP and an ATP-regenerating system

Calcium loading of a rat parotid microsomal fraction is greatly increased by an ATP-regenerating system (phosphocreatine and creatine phosphokinase). This effect is neither a consequence of a rise in the ATP concentration nor of an increased formation of inorganic phosphate originating from hydrolysis of ATP or phosphocreatine. Addition of ADP to the incubation medium provokes an inhibition of Ca2+ influx and a stimulation of Ca2+ efflux by the microsomal fraction. These results suggest that the stimulation of Ca2+ uptake by the ATP-regenerating system is due, at least in part, to an increase of Ca2+ influx and a slowing down of Ca2+ efflux as consequence of a decrease of ADP availability. It is proposed that the effect of ADP on Ca2+ movements could account for the action of certain agonists on intracellular Ca2+ concentration. Moreover, the InsP3 responsive Ca2+ pool was also shown to be enlarged by the ATP-regenerating system without modification of InsP3 sensitivity.

Membrane translocation and regulation of uridine diphosphate-glucuronic acid uptake in rat liver microsomal vesicles

Background/Aims : Hepatic glucuronidation is quantitatively the most important conjugation reaction by which an array of endogenous compounds and xenobiotics undergo biotransformation and detoxification. The active site of the uridine diphosphate (UDP) glucuronosyltranferases, which catalyze glucuronidation reactions, has been postulated to reside in the lumen of the endoplasmic reticulum. The aim of this study was to characterize the process whereby UDP glucuronic acid (UDP-GIcUA), the cosubstrate for all glucuronidation reactions, is transported into microsomal vesicles. Methods : The uptake process was analyzed using rapid filtration techniques, radiolabeled UDP-GIcUA, and rat liver microsomes. Results : Uptake was saturable with respect to time and concentration, inhibited by 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid and 4-acetamido-4′-isothio-cyanatostilbene-2-2′-disulfonic acid, and was osmotically sensitive. Transport was stimulated by Mg 2+ and guanosine triphosphate (50 μmol/L) but not guanosine 5′-O-(3-thiotriphosphate) or adenosine triphosphate. Luminal UDP- N-acetylglucosamine (1 mmol/L) produced enhanced uptake of UDP-GIcUA ( trans stimulation). In contrast to nucleotide sugar transport in the Golgi apparatus, trans uridine monophosphate and UDP did not alter UDP-GIcUA transport in microsomes, indicating distinct processes. Conclusions : These data provide unambiguous evidence for the existence of a unique, substrate-specific, regulated, carrier-mediated process that transports UDP-GIcUA into the lumen of hepatocyte microsomes. This transporter may regulate glucuronidation in vivo.

Murine pulmonary Ca(2+)-transport system activated by allergic immune response retains sensitivity to oxidative stress.

Exaggerated oxygen radical production by airway cells may contribute to increased airway responsiveness and heightened smooth muscle constriction in asthmatic lungs. Smooth muscle cell contractility in the lung is regulated by Ca2+ homeostasis. The contribution of inflammatory cells to these events is unclear. A murine model of allergic pulmonary hypersensitivity was developed to study the role of Ca2+ transport in allergic pulmonary reactions. Sensitization of mice was accomplished by injection with ovalbumin (OA) (1 or 50 micrograms) or OA (1 microgram) plus Al(OH)3. Pulmonary responses were elicited by inhalation provocation challenge with OA aerosol and quantified by the extent of inflammatory cell infiltrate at 24 h. Increased Ca2+ transport was found in microsomes and homogenates of the lung after antigen challenge. Activation of Ca2+ transport was correlated with the severity of the allergic pulmonary response as evidenced from specific antibody production and inflammatory cell infiltrate. The greatest increase in Ca2+ transport was noted in microsomes from mice sensitized with OA plus adjuvant. Ca2+ transport in sensitized, but not in control mice, was responsive to oxidative stress induced by addition of phenol and hydrogen peroxide. Lung homogenates from both groups of animals responded similarly to phenoxyl radical-induced oxidative stress induced by phenol plus exogenous tyrosinase. These results are the first to indicate heightened Ca2+ transport in pulmonary microsomes following an allergic lung response and emphasize the role of aluminum hydroxide in enhancing allergic reactions in the lung. The responsiveness of the system to oxidative stress suggests that oxidative mechanisms may contribute to the physiologic and pathologic manifestations, such as airway hyperreactivity, associated with allergic pulmonary disease.

Formation and Reduction of Ascorbate Radicals by Hog Thyroid Microsomes

The formation of ascorbate radicals, identified by ESR experiments, was observed in the ascorbate peroxidase reaction by thyroid microsomes. The steady-state concentration of ascorbate radicals decreased in the presence of NADH. The oxidation of NADH was followed optically. Using the ascorbic acid oxidase system, NADH-dependent electron transport in thyroid microsomes was examined. Ascorbate radicals competed with bound cytochrome b 5 for the reaction with reduced NADH-cytochrome b 5 reductase. The NADH-ascorbate radical reductase activity of thyroid microsomes was calculated to be 0.17 μmol/mg · s at 3.3 μM ascorbate radicals. Kinetic results show that the properties of NADH-cytochrome b 5 reductase in thyroid microsomes were similar to those of the enzyme in liver microsomes. The formation of ascorbate radicals by thyroid microsomes was stimulated by the addition of thyroxine, and the stimulation was decreased also by NADH. The thyroxine-mediated oxidation of ascorbate is explained in terms of consecutive one-electron transfers initiated by bound thyroid peroxidase. These results, along with those described in our previous paper (M. Nakamura, I. Yamazaki, and S. Ohtaki, 1990, J. Biochem. 108, 804-810), support the idea that ascorbate protects thyroid hormones from oxidative degradation through the NADH-cytochrome b 5 reductase system.

Characterization of ATP-dependent proton transport in medullary bone-derived microsomes

Proton transport in microsomal vesicles derived from medullary bone of laying hens was observed to be inhibited in a dose-dependent manner with fusidic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diatzole (NBD-Cl), duramycin and dicyclohexylcarbodiimide (DCCD). The IC50 values were 570 microM, 4.5 microM, 10 micrograms/ml and 32 microM for fusidic acid, NBD-Cl, duramycin and DCCD, respectively. 14C-DCCD labeled a single protein band of 15-17 kDa from bone-derived microsomes in SDS-electrophoresis. A protein of this size is a proton-conducting subunit of the vacuolar ATPases. Further, the proton transport was found to be electrogenic, thus it generates the membrane potential across the vesicle membrane. The generation of membrane potential was inhibited using 100 nM bafilomycin A1, which in low concentrations is a specific inhibitor of vacuolar ATPases. The presence of Cl- was essential for maximal proton transport activity. These results confirm the electrogenicity and extend the characterization of the osteoclastic H(+)-ATPase.

Regulation of hepatic microsomal glucose transport.

Regulation of hepatic microsomal glucose transport.

NADPH-dependent inhibition of lipid peroxidation in rat liver microsomes

Microsomal NADPH-driven electron transport is known to initiate lipid peroxidation by activating oxygen in the presence of iron. This pro-oxidant effect can mask an antioxidant function of NADPH-driven electron transport in microsomes via vitamin E recycling from its phenoxyl radicals formed in the course of peroxidation. To test this hypothesis we studied the effects of NADPH on the endogenous vitamin E content and lipid peroxidation induced in liver microsomes by an oxidation system independent of iron: an azo-initiator of peroxyl radicals, 2,2'-azobis (2,4-dimethylvaleronitrile), (AMVN), in the presence of an iron chelator deferoxamine. We found that under conditions NADPH: (i) inhibited lipid peroxidation; (ii) this inhibitory effect was less pronounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats; (iii) protected vitamin E from oxidative destruction; (iv) reduced chromanoxyl radicals of vitamin E homologue with a 6-carbon side-chain, chromanol-alpha-C-6. Thus NADPH-driven electron transport may function both to initiate and/or inhibit lipid peroxidation in microsomes depending on the availability of transition metal catalysts.

Glycogen Storage Disease Diagnosed in Adults

Glycogen storage diseases are usually identified in childhood. We present the clinical, biochemical and histological features of 10 patients first diagnosed in adult life. Five had glycogen storage disease type 1a, one type 1c, two type IX, and in two patients there were previously unreported abnormalities of hepatic glucose-6-phosphatase system activity. Of the latter, one patient had an inhibitor of liver glucose-6-phosphatase (pseudo-1b glycogen storage disease) the other having abnormal glucose-6-phosphatase activity and microsomal pyrophosphate transport. A glucagon test is suggested as a useful screening procedure. Glycogen storage disease should be considered in adults with symptoms suggesting hypoglycaemia.

ドナー肺保存時のミクロソーム電子伝達系の障害： 保存温度・時間相関ノモグラムの作製

ドナー肺保存時のミクロソーム電子伝達系の障害： 保存温度・時間相関ノモグラムの作製

Effect of cadmium on Ca 2+ transport in brain microsomes

The effect of Cd 2+ on Ca 2+ transport properties (uptake/release) in rat brain microsomes is examined by the tracer method using 45Ca 2+ Cadmium ion (Cd 2+) shows a dose-dependent inhibition of Ca 2+-ATPase activity and consequently, exhibits a reduction in ATP-dependent Ca 2+ uptake. In addition to this, Cd 2+ also stimulates a rapid release of Ca 2+ ( t 1 2 = 0.5 min ) from the microsomes in a dose-dependent manner. The effect of Cd 2+ is reversible by 1 mM cysteine or dithiothreitol (DTT). It is suggested that Cd 2+ plays an important role in regulating the transmembrane flux of the cations in the microsomes. This effect is dramatically modulated by DTT suggesting a role of sulfhydryl groups in Ca 2+ -transport.

Co-expression of slow-twitch/cardiac muscle Ca2+-ATPase (SERCA2) and phospholamban

Full length cDNAs encoding both slow-twitch/cardiac (SERCA2) and fast-twitch skeletal muscle (SERCA1) Ca2+-ATPases were expressed by transient transfection of COS-1 cells. Studies of the Ca2+-dependency of Ca2+-transport in microsomes isolated from these cells showed that both isoforms had an affinity for Ca2+ of about 0.2 μM. The Ca2+-affinity of SERCA2 was lowered when phospholamban was co-expressed with it, demonstrating that the two proteins interact in this expression system. These studies support the view that phospholamban inhibition accounts for the low Ca2+-amnity and low activity of SERCA2 in cardiac muscle sarcoplasmic reticulum.

Antioxidant effects of ubiquinones in microsomes and mitochondria are mediated by tocopherol recycling

Ubiquinones and tocopherols (vitamin E) are intrinsic lipid components which have a stabilizing function in many membranes attributed to their antioxidant activity. The antioxidant effects of tocopherols are due to direct radical scavenging. Although ubiquinones also exert antioxidant properties the specific molecular mechanisms of their antioxidant activity may be due to: (i) direct reaction with lipid radicals or (ii) interaction with chromanoxyl radicals resulting in regeneration of vitamin E. Lipid peroxidation results have now shown that tocopherols are much stronger membrane antioxidants than naturally occurring ubiqinols (ubiquinones). Thus direct radical scavenging effects of ubiquinols (ubiquinones) might be negligible in the presence of comparable or higher concentrations of tocopherols. In support of this our ESR findings show that ubiquinones synergistically enhance enzymic NADH- and NADPH-dependent recycling of tocopherols by electron transport in mitochondria and microsomes. If ubiquinols were direct radical scavengers their consumption would be expected. Further proving our conclusion HPLC measurements demonstrated that ubiquinone-dependent sparing of tocopherols was not accompanied by ubiquinone consumption.