Liver Endoplasmic Reticulum Research Articles

A glycoprotein from rat liver endoplasmic reticulum promotes both aggregation and fusion of liposomes at acidic pH.

Low-pH-induced fusion of liposomes with rat liver endoplasmic reticulum was evidenced. Fusion was inactivated by treatment of microsomes with trypsin or EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline), indicating the involvement of a protein. The protein was purified 555-fold by chromatographic steps. The identification and purification to homogeneity was obtained by electroelution from a slab gel, which gave a still active protein of about 50 kDa. The protein promoted the fusion of liposomes; laser light scattering showed an increase of mean radius of vesicles from 60 up to about 340 nm. Fusion was studied as mass action kinetics, describing the overall fusion as a two-step sequence of a second order aggregation followed by a first order fusion of liposomes. For phosphatidylcholine containing liposomes aggregation was not rate-limiting at pH 5.0 and fusion followed first order kinetics with a rate constant of 13 . 10(-3) sec-1. For phosphatidylethanolamine/phosphatidic acid liposomes aggregation was rate-limiting; however, the overall fusion was first order process, suggesting that fusogenic protein influences both aggregation and fusion of liposomes. The protein binds to the lipid bilayer of liposomes, independently of pH, probably by a hydrophobic segment. Exposed carboxylic groups might be able to trigger pH-dependent aggregation and fusion. It is proposed that the protein inserted in the lipid bilayer bridges with an adjacent liposome forming a fused doublet. Since at endoplasmic reticulum level proton pumps are operating to generate a low-pH environment, the membrane bound fusogenic protein may be responsible for both aggregation and fusion of neighboring membranes and therefore could operate in the exchange of lipidic material between intracellular membranes.

Positive feedback between ethanolamine-specific phospholipid base exchange and cytochrome P450 activities in rat liver microsomes. The effect of clofibric acid

The results of the present investigation relate the effects of the nutritional state and administration of clofibric acid (CLA), a hypolipidaemic drug and peroxisomal proliferator, on phosphatidylethanolamine (PE) synthesis in rat liver and fatty acid metabolism. Fasting and CLA treatment of animals causes an increase in the amount of PE in endoplasmic reticulum (ER) membranes and mitochondria, as well as in the PE/phosphatidylcholine (PC) ratio. Moreover, the activity of the ethanolamine-specific phospholipid base exchange (PLBE) enzyme in liver ER membranes of fasted animals was enhanced by 75% in comparison to that of animals fed ad libitum. The effect of CLA treatment was additive to that of starvation; PE synthesis tested in vitro via the Ca 2+-sensitive PLBE reaction increased 3-fold in comparison to rats fed ad libitum. This is confirmed by an increased V max for the reaction, but the affinity of the enzyme for ethanolamine was not significantly changed. These effects were accompanied by an enhanced expression of cytochrome P450 CYP4A1 isoform and elevated activity of the enzyme upon CLA administration. The stimulatory effect of CLA administration on the efficiency of the ethanolamine-specific PLBE reaction can be explained by elimination of lauric acid, a known inhibitor of de novo PE synthesis, during the course of ω-hydroxylation catalysed by CYP4A1, and by increased expression of the PLBE enzyme. The products of ω-hydroxylation of lauric acid, which are then converted by dehydrogenase to 1,12-dodecanedioic acid, did not significantly affect the in vitro synthesis of PE.

Identification of endoplasmic reticulum proteins involved in glycan assembly: synthesis and characterization of P3-(4-azidoanilido)uridine 5'-triphosphate, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins.

Much of the enzymic machinery required for the assembly of cell surface carbohydrates is located in the endoplasmic reticulum (ER) of eukaryotic cells. Structural information on these proteins is limited and the identity of the active polypeptide(s) is generally unknown. This paper describes the synthesis and characteristics of a photoaffinity reagent that can be used to identify and analyse members of the ER glycan assembly apparatus, specifically those glycosyltransferases, nucleotide phosphatases and nucleotide-sugar transporters that recognize uridine nucleotides or UDP-sugars. The photoaffinity reagent, P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP), was synthesized easily from commercially available precursors. AAUTP inhibited the activity of ER glycosyltransferases that utilize UDP-GlcNAc and UDP-Glc, indicating that it is recognized by UDP-sugar-binding proteins. In preliminary tests AAUTP[alpha-32P] labelled bovine milk galactosyltransferase, a model UDP-sugar-utilizing enzyme, in a UV-light-dependent, competitive and saturable manner. When incubated with rat liver ER vesicles, AAUTP[alpha-32P] labelled a discrete subset of ER proteins; labelling was light-dependent and metal ion-specific. Photolabelling of intact ER vesicles with AAUTP[alpha-32P] caused selective incorporation of radioactivity into proteins with cytoplasmically disposed binding sites; UDP-Glc:glycoprotein glucosyltransferase, a lumenal protein, was labelled only when the vesicle membrane was disrupted. These data indicate that AAUTP is a membrane topological probe of catalytic sites in target proteins. Strategies for using AAUTP to identify and study novel ER proteins involved in glycan assembly are discussed.

P2A40 - Different inactivation of NADPH- and NADH-dependent chains of rat liver endoplasmic reticulum in tetrachloromethane

P2A40 - Different inactivation of NADPH- and NADH-dependent chains of rat liver endoplasmic reticulum in tetrachloromethane

Purification and Characterization of Hamster Liver Microsomal 7α-Hydroxycholesterol Dehydrogenase: SIMILARITY TO TYPE I 11β-HYDROXYSTEROID DEHYDROGENASE

While studying the bile acid synthetic pathway of hamsters, we discovered an NADP+-dependent liver microsomal 7alpha-hydroxycholesterol dehydrogenase (7alpha-HCD) activity that was not observed in rat liver microsomal fractions. The hamster liver microsomal 7alpha-HCD was purified to homogeneity using 2', 5'-ADP and cholic acid-agarose affinity chromatography. 7alpha-HCD displayed a molecular weight of approximately 34,000 on SDS-polyacrylamide gel electrophoresis; it is an intrinsic membrane protein of the hamster liver endoplasmic reticulum and exists as a multimeric aggregate in pure form. Partial N-terminal amino acid sequence analysis showed that 7alpha-HCD had high sequence similarity to human 11beta-hydroxysteroid dehydrogenase (11beta-HSD; 24/30 amino acid identity). The Km values for corticosterone and 7alpha-hydroxycholesterol were 1.2 and 1.9 microM, respectively, for purified 7alpha-HCD; both reactions displayed identical Vmax values (approximately 170 nmol/min/mg of protein). The IC50 of carbenoxolone, a competitive inhibitor of 11beta-HSD, was 75 nM for 7alpha-hydroxycholesterol dehydrogenation and 210 nM for corticosterone dehydrogenation. The tissue-specific expression in hamster was as follows: adrenal >/= liver > kidney > testis >> brain > lung. Microsomal 7alpha-HCD is uniquely expressed in hamster liver and to some extent in human liver but not in rat liver. Western blot analysis with two antibodies elicited against an N-terminal peptide of the human 11beta-HSD and purified hamster liver 7alpha-HCD, respectively, suggested the presence of multiple forms of 7alpha-HCD in hamster liver, most likely due to the existence of a family of 11beta-HSD proteins. Since 7-oxocholesterol is a potent inhibitor of cholesterol 7alpha-hydroxylase, alternative mechanisms for regulation of bile acid synthesis may exist in human and hamster liver due to production of this metabolite and its potential as an oxysterol.

Microsomal steroid sulfatase: interactions with cytosolic steroid sulfotransferases

Net sulfation of 4-methylumbelliferone in intact hepatocytes is regulated, in part, by substrate cycling between sulfotransferases (SULT) and arylsulfatases (ARS). Thus, ARS have the potential to influence rates of net sulfate conjugation of a variety of compounds in intact cells via interaction with SULT. Unlike ARSA and ARSB, which are lysosomal, steroid sulfate sulfatase (ARSC, also known as STS) is localized exclusively in the endoplasmic reticulum (ER). The present study was designed to assess the existence and extent of substrate cycling between steroids and their sulfate conjugates through ARSC and SULT, and also to initiate studies of the topology of the catalytic site of ARSC in the rat liver ER. Addition of rat liver microsomes to cytosol and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) reduced rates of sulfation of dehydroepiandrosterone (DHEA) by SULT, and similarly hydrolysis of DHEA sulfate (DHEAS) was reduced when recombinant human hydroxysteroid SULT was added to rat liver microsomes in the presence of PAPS. There was no evidence for ARSC latency in the presence of detergent at either 4 or 37°C, indicating that facilitated transport of steroid sulfates across the ER membrane may not be required for ARSC activity. The effect of proteases on ARSC activity in intact and disrupted microsomes was determined and compared with effects on components of the glucose-6-phosphatase system known to be localized on the lumenal and cytoplasmic surfaces of the ER. In contrast to the components of the glucose-6-phosphatase system, activity of ARSC in both intact and disrupted microsomes was substantially more resistant to protease inactivation. Our results indicate that substrate cycling of steroids and their sulfates does occur, and suggest that the active site of ARSC may be located within the ER membrane.

A stress-inducible rat liver endoplasmic reticulum protein, ERp29.

We have isolated, cDNA cloned and characterised a 29-kDa protein (ERp29), containing a C-terminal endoplasmic reticulum(ER)-retrieval signal, from the rat liver ER. ERp29 was induced to high levels in the rat hepatoma cells under metabolic stress conditions known to cause an aberrant accumulation of proteins in the ER [(e.g. culture in presence of the Ca2+ ionophore A23187, inhibitors of Ca2+-ATPase (thapsigargin), intracellular protein transport (brefeldin A), or protein N-glycosylation (tunicamycin)]. Experimental evidence of its localisation in the luminal compartment of the ER was obtained by topology studies including immunofluorescence microscopy, in vitro translation and proteinase protection assay. ERp29 constitutes about 0.1% of the rat hepatic microsomal proteins and is constitutively expressed in all rat tissues examined, as evident from northern blot analysis. In rat hepatoma cells ERp29 was found to be associated with the abundant molecular chaperone/stress protein BiP/GRP78 and this interaction was significantly enhanced after treatment with tunicamycin and A23187. Taken together, these results suggest that ERp29 is a member of the stress-response machinery of the ER.

A functional role for histidyl residues of the UDP-glucuronic acid carrier in rat liver endoplasmic reticulum membranes.

Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). To determine the crucial amino acids of the membrane transporter and evaluate their function in regulating the glucuronidation reaction, we examined the effect of histidyl-specific irreversible inhibitors on the uptake of radiolabeled UDP-GlcUA in rat liver ER. Inactivation of uptake (initial rate) was more pronounced with hydrophobic reagents [diethyl pyrocarbonate (DEPC), p-bromophenacyl bromide] as compared to the more hydrophilic reagent (p-nitrobenzenesulfonic acid methyl ester). DEPC was used to further characterize the inhibition because of its greater specificity for protein histidyl residues. While initial [14C]UDP-GlcUA uptake rates were diminished by DEPC treatment of intact microsomes, the accumulation of isotope at equilibrium was not significantly affected, indicating no loss of vesicle integrity. A pKa of approximately 7 for the modified residue(s) of the transporter supported the alkylation of imidazole moieties. Protection against inactivation was observed with UDP-GlcUA as well as other nucleotide-sugars known for their interaction with this transporter. Uptake activity of the transporter (Vmax) but not UDP-GlcUA binding (Km) was affected by a limited inactivation. Furthermore, a partial inactivation of the transporter impaired the binding of the photoaffinity label [beta-32P]5-azido-UDP-GlcUA to UDP-glucuronosyltransferases (UGTs) in intact, but not in detergent-disrupted, ER vesicles. These results demonstrate the involvement of histidyl residue(s) in the UDP-GlcUA uptake process in rat liver ER, provide additional evidence for the lumenal orientation of the UGT active site, and support the view that translocation of the UGT cosubstrate is a rate-limiting step of the glucuronidation reaction.

Interaction of caldesmon with endoplasmic reticulum membrane: effects on the mobility of phospholipids in the membrane and on the phosphatidylserine base-exchange reaction.

We have previously demonstrated by tryptophan fluorescence the interaction of caldesmon with anionic phospholipid vesicles [Czurylo, Zborowski and Dabrowska (1993) Biochem. J. 291, 403-408]. In the present work we investigated the interaction of caldesmon with natural-membrane (rat liver endoplasmic reticulum) phospholipids by co-sedimentation assay. The results indicate that 1 mol of caldesmon binds approx. 170 mol of membrane phospholipids with a binding affinity constant of 7.3 x 10(6) M-1. The caldesmon-membrane phospholipid complex dissociates with increasing salt concentration and in the presence of Ca2+/calmodulin. As indicated by EPR measurements of membrane lipids labelled with 5-doxyl stearate and TEMPO-phosphatidylethanolamine, binding of caldesmon results in an increase in mobility of the acyl chains (in the region of carbon 5) and a decrease in polar headgroup mobility of phospholipids. Interaction of caldesmon with phospholipids is accompanied by inhibition of phosphatidylethanolamine synthesis via a phospholipid base-exchange reaction, with phosphatidylserine as substrate. This shows that, of the endoplasmic reticulum membrane phospholipids, the main target of caldesmon is phosphatidylserine.

Apolipoprotein B Is Intracellularly Associated with an ER-60 Protease Homologue in HepG2 Cells

Two ALLN (N-acetyl-leucyl-leucyl-norleucinal)-sensitive endoplasmic reticulum (ER)-localized proteases (ER-60 and ER-72) were recently purified from rat liver. We used an antibody to rat ER-60 to investigate the possible role of this protease in apolipoprotein B (apoB) degradation. First, immunoprecipitation and immunoblotting experiments with the anti-rat ER-60 antibody suggested that HepG2 cells contain a homologue of ER-60 with an approximate molecular mass of 58-60 kDa. The ER-60 homologue was mostly associated with the luminal contents of HepG2 microsomes. Evidence from co-immunoprecipitation and cross-linking experiments appear to suggest that the ER-60 homologue in HepG2 cells is associated with apoB intracellularly. A small pool of apoB was recovered when HepG2 lysates were subjected to immunoprecipitation with anti-rat ER-60 antibody followed by a second immunoprecipitation with anti-apoB antibody. Furthermore, cross-linking of permeabilized cells with dithiobis(succinimidylpropionate) further demonstrated association of apoB with the ER-60 homologue in HepG2 cells. Three polypeptides with molecular masses of 78, 66, and 50 kDa were consistently found to be associated with apoB as well as the 58-kDa ER-60 homologue. The 78-kDa protein associated with both apoB and ER-60 appeared to represent immunoglobulin heavy chain-binding protein (BiP) based on immunoprecipitation with a monoclonal antibody. Cross-linking and immunoblotting experiments suggested the association of the 78-kDa BiP with both the 58-kDa ER-60 homologue as well as the 550-kDa apoB. In summary, the data suggests that HepG2 cells contain a 58-kDa protein which is homologous to the rat liver ER-60 in size, antigenecity, and intracellular localization. The ER-60 homologue in HepG2 cells appears to be closely associated with apoB, as well as other proteins possibly representing ER chaperones such as BiP. We hypothesize that the ER-60 homologue may be involved in the degradation of apoB in the ER lumen of HepG2 cells.

An antagonist of ATP-regulated potassium channels, the guanidine derivative U-37883A, stimulates the synthesis of phosphatidylserine in rat liver endoplasmic reticulum membranes

The guanidine derivative U-37883A has been found to stimulate in vitro synthesis of phosphatidylserine in endoplasmic reticulum membranes, catalyzed exclusively by a serine-specific base exchange enzyme. The stimulation of the enzyme activity by the drug was concentration-dependent, with EC 50 of 54 μM, while the biologically inactive analog of U-37883A, U-42069, was without effect. The stimulation caused by U-37883A was enhanced under the conditions when active transport of Ca 2+ into the lumen of microsomal vesicles was induced, whereas it was inhibited by a calcium ionophore, A23187, and by a specific inhibitor of Ca 2+-ATPase, thapsigargin. On the other hand, a potassium ionophore, valinomycin, had no effect on phosphatidylserine synthesis. U-37883A did not affect the K m of the base exchange enzyme for serine, but greatly reduced the EC 50 value of the enzyme for calcium. Furthermore, Ca 2+ uptake by endoplasmic reticulum vesicles has been found to increase in the presence of U-37883A. These observations suggest that U-37883A enhances phosphatidylserine synthesis indirectly by acting on calcium transport, thus affecting calcium concentration within the lumen of endoplasmic reticulum membranes. Alternatively, the effect of the drug could be propagated via the mechanism by which phospholipid flip-flop movement, known to regulate the serine-specific base exchange reaction, is modulated.

Changes of adenosine triphosphate-dependent calcium uptake in microsomal fractions of rat liver during sepsis

Background. Intracellular calcium concentration is an important regulator of cellular metabolism. Endoplasmic reticulum membranes play an important role in the regulation of cytoplasmic calcium in the mammalian liver. The characterization of the changes of calcium uptake in endoplasmic reticulum may contribute to the potential intracellular mechanisms for cellular dysfunction during sepsis. Methods. The effects of sepsis on the calcium uptake in rough endoplasmic reticulum of rat liver were studied. Sepsis was induced by means of cecal ligation and puncture (CLP). The control rats underwent sham operation. Microsomal fractions were isolated from the liver with differential centrifugation. Results. The calcium uptake by liver endoplasmic reticulum was decreased by 30% to 35% (p < 0.05) during early sepsis (9 hours after CLP) and by 38% to 43% (p < 0.05) during late sepsis (18 hours after CLP), respectively. The maximum velocity values for adenosine triphosphate (ATP) and for Ca 2+ were also decreased by 25% to 37% (p < 0.05) during early sepsis and by 35% to 42% (p < 0.05) during late sepsis. The Michaelis-Menten constant for ATP and Ca 2+ transport had no difference among three groups. The magnesium stimulation and vanadate inhibitory activity were also decreased by 17% to 38% (p < 0.05) during early sepsis and by 34% to 50% (p < 0.05) during late sepsis. Conclusions. These data demonstrate that ATP-dependent calcium uptake in rough endoplasmic reticulum of rat liver was impaired during early and late sepsis. Because the low intracellular calcium concentration plays an important role in the regulation of cellular function, an impairment in the ATP-dependent calcium uptake by endoplasmic reticulum during early and late sepsis may have a pathophysiologic significance in contributing to the development of altered hepatic metabolism during sepsis.

Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver.

Prompted by previous observations that polymannose oligosaccharides are released from newly synthesized glycoproteins [Anumula and Spiro (1983) J. Biol. Chem. 258, 15274-15282], we examined rat liver endoplasmic reticulum (ER) for the presence of endoglycosidases that could be involved in an event presumed to be a function of the protein quality control machinery. Our investigations indicated that a peptide:N-glycanase (PNGase) is present in ER membranes that has the capacity to release from radiolabelled glycopeptides glucosylated as well as non-glucosylated polymannose oligosaccharides terminating at their reducing end in a di-N-acetylchitobiose sequence (OS-GlcNAc2). This enzyme, which was found to be luminal in orientation, was most active in the pH range 5.5-7.0 and although it had no exogenous bivalent-cation requirements it was inhibited by EDTA. Detailed studies with Man9GlcNAc2-peptides demonstrated that in addition to the free oligosaccharide (Man9GlcNAc2) an additional neutral product characterized as Man9GlcNAc2 linked to an as yet unidentified aglycone was released in a manner that suggests its role as an intermediate. Our observation that ER, in contrast with cytosol, had no endo-beta-N-acetylglucosaminidase activity would indicate that oligosaccharides terminating in a single GlcNAc residue (OS-GlcNAc1), which have been noted to appear in the extravesicular compartment shortly after N-glycosylation [Moore and Spiro (1994) J. Biol. Chem. 269, 12715-12721] are released from the protein as OS-GlcNAc2 and undergo an ER-to-cytosol translocation in that form before undergoing cleavage of their chitobiose core.

Two kinetically-distinct components of UDP-glucuronic acid transport in rat liver endoplasmic reticulum

Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-G1cUA) in rat liver endoplasmic reticulum (ER). Measurement of uptake at varying concentrations of high specific activity [β- 32P]UDP-G1cUA has revealed the presence of a two component UDP-G1cUA transporting system. Transport at low substrate concentrations occurred predominantly via a high affinity component ( K m = 1.6 μM), whereas a low affinity component ( K m = 38 μM) predominated at high substrate concentrations. The K m for the high affinity system is in agreement with that previously published, while the low affinity component is a new finding. The uptake of UDP-G1cUA was temperature-sensitive, time dependent, and saturable for both components. The high affinity transport was affected by trans-stimulation and cis-inhibition by UDP- N-acetylglucosamine (UDP-G1cNAc); however, the same concentrations of UDP-G1cNAc had less effect on the low affinity system. In order to further study the two transport components, various inhibitors of anion transport carriers were tested. The high affinity component was strongly inhibited by 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) and furosemide, while the low affinity system was less sensitive to these reagents. Dose-dependent inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) was found for both transport systems. Probenecid was found to be a weak inhibitor of both components of the UDP-G1cUA uptake. Finally, the major metabolite of 3′-azido-3′-deoxythymidine, 3′-azido-3′-deoxythymidine monophosphate (AZTMP), was able to inhibit the uptake of UDP-GlcUA by both components. The results indicate the presence of two carrier-mediated UDP-glucuronic acid transporting components in rat liver ER.

Endoplasmic reticulum phosphate transport

The major role of the liver endoplasmic reticulum phosphate/pyrophosphate transport proteins is the regulation of blood glucose levels. The glucose-6-phosphatase enzyme is an endoplasmic reticulum enzyme system which hydrolyzes glucose-6-phosphate to glucose and phosphate. Glucose-6-phosphatase is the terminal step of both gluconeogenesis and glycogenolysis. The glucose-6-phosphatase enzyme is a very hydrophobic membrane protein and its active site is inside the lumen of the endoplasmic reticulum. The substrates and products of the enzyme therefore have to cross the endoplasmic reticulum membrane. The glucose-6-phosphatase enzyme is associated with a calcium binding protein (SP). There are also transport proteins for the substrate glucose-6-phosphate (T1) and the products phosphate (T2) and glucose (T3). There appear to be at least two different liver endoplasmic reticulum proteins that can transport phosphate. One of the proteins T2b can also transport pyrophosphate and carbamyl phosphate which are also substrates for the glucose-6-phosphatase enzyme. The metabolic regulation, genetic deficiencies, ontogeny and tissue distribution of the endoplasmic reticulum T2 proteins will be described.

Permeability of Liver Microsomal Membranes to Glucose

The permeability of rat liver microsomes to glucose has been studied by using14C-labelled D-glucose and a light-scattering technique.1) The microsomal intravesicular apparent isotope space for D-glucose (1mM; after 5min incubation at 22°C) was 2.34 μl/mg protein, i.e., approximately 72% of the apparent water space.2) Efflux of [14C]D-glucose from microsomal vesicles pre-loaded as in 1) and measured by rapid Millipore filtration after dilution (100 fold) in a glucose-free medium revealed that 15 sec after dilution only 15% of intravesicular glucose was still retained by microsomes.3) Osmotic behaviour of microsomes upon addition of D-glucose measured by a light-scattering technique revealed a glucose influx, saturable at [D-glucose] ≥ 100 mM, and (partially) inhibited by pentamidine and cytochalasin B. Ascorbic acid, L-glucose and other monosaccharides and related compounds also permeated liver microsomes in a fashion similar to D-glucose. These data indicate the existence of a facilitative transport system(s) for glucose in the membrane of liver endoplasmic reticulum vesicles.

EFFECT OF CHRONIC ETHANOL CONSUMPTION ON GLYCOSYLATION PROCESSES IN RAT LIVER MICROSOMES AND GOLGI APPARATUS

Previous studies have demonstrated that acute ethanol intoxication affects various steps of protein glycosylation at the level of rat liver endoplasmic reticulum and Golgi apparatus. The aim of this investigation was to demonstrate whether chronic ethanol intake can induce definitive changes of liver glycoprotein processing. Rats were given ethanol by liquid diet for 8 weeks. At the end of this period the triglyceride levels in liver homogenate and microsomes were significantly higher than in controls. Isolated hepatocytes prelabelled with [3H]Na palmitate and [14C]glucosamine showed a significant storage of the lipid and carbohydrate radioactivity in microsomes and Golgi apparatus and a significant impairment of labelled glycolipoprotein secretion. Changes of the glycosylation steps were observed both in endoplasmic reticulum and in Golgi apparatus: in the former the levels of dolichyl phosphate, which is rate-limiting for the synthesis of glycoprotein, showed a significant reduction; in the latter the activity of the main enzymes responsible for the terminal glycosylation process was significantly decreased. These data suggest that an impairment of glycoprotein maturation may be involved in the pathogenesis of liver injury induced by chronic ethanol intake.

Effects of 1,2-dichloroethane intoxication on dolichol levels and glycosyltransferase activities in rat liver microsomes and Golgi apparatus

Rat intoxication with a single dose of 1,2-dichloroethane (DCE) (50 μl/100 g b.w) is able to induce a significant modification of protein glycosylation in the liver endoplasmic reticulum and Golgi apparatus. HPLC analysis shows that within 5–60 min after DCE-intoxication, the levels of total dolichol, free dolichol and dolichyl phosphate strongly decreased in the microsomes and Golgi apparatus. Particularly in total microsomes, dolichyl phosphate, which is ratelimiting for the biosynthesis of the N-linked oligosaccharide chains, drops to values significantly lower than in the control group 15 min after DCE poisoning. In the Golgi apparatus, the total dolichol, essential to enhance the fluidity and permeability of these membranes, early and significantly decreases already 5 min after DCE poisoning. Moreover, in the Golgi apparatus galactosyl- and sialyltransferase activities, the main enzymatic activities of terminal protein glycosylation, are significantly reduced, as measured 15 min after DCE intoxication. These data suggest that the impairment of glycoprotein synthesis, maturation and secretion may be involved in the pathogenesis of liver injury induced by acute DCE-intoxication.

Characterization of N-ethylmaleimide-sensitive thiol groups required for the GTP-dependent fusion of endoplasmic reticulum membranes.

The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required for the structural maintenance and post-mitotic regeneration of the endoplasmic reticulum. This fusion is sensitive to the thiol-alkylating agent N-ethylmaleimide. In many intracellular fusion events N-ethylmaleimide-sensitivity is associated with a homotrimeric ATPase called N-ethylmaleimide-sensitive fusion protein or NSF. The addition of cytosol containing NSF is known to restore fusion activity to N-ethylmaleimide-treated membranes. We found that the inhibition of fusion of rat liver endoplasmic reticulum membranes (microsomes) by N-ethylmaleimide was not reversed by the addition of untreated cytosol. Fusion was also unaffected by treatment with a buffer known to remove NSF from membranes. Accordingly, no membrane-associated NSF was detected by immunoblot analysis. These data suggest that microsome fusion requires an N-ethylmaleimide-sensitive component distinct from NSF. This component was tightly associated with the membranes, so we used a number of chemical probes to characterize it in situ. Its thiol groups did not appear to be part of a GTP-binding site. They showed relatively low reactivity with sodium periodate, which induces the formation of disulphide bonds between proximate thiol groups. The thiols were not protected against N-ethylmaleimide by Zn2+, a potent inhibitor of fusion which is known to efficiently co-ordinate thiol groups. To characterize the topology of the fusion-related thiol groups we used bulky thiol-specific reagents prepared by conjugating BSA or 10 kDa aminodextran to the bifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibition of fusion by these reagents indicated that these thiols are highly exposed on the membranes. This exposure might be important for the function of these groups during GTP-triggered fusion.

CDNA Cloning and Baculovirus Expression of the Human Liver Endoplasmic Reticulum P58: Characterization as a Protein Disulfide Isomerase Isoform, but Not as a Protease or a Carnitine Acyltransferase

The function of a 58-kDa liver microsomal protein (P58) is controversial. To help clarify the physiological function of this protein, particularly in humans, a full-length human liver cDNA clone was isolated, sequenced, and expressed in milligram quantities with the use of a baculovirus expression system. The deduced amino acid sequence of the mature protein contained two thioredoxin-like active site motifs (CGHC) and in its C-terminus a nuclear localization motif (KPKKKKK), and an ER-retention/retrieval motif (QEDL). The mature form of human P58 shared 95% amino acid sequence identity with the deduced amino acid sequences of a bovine liver cDNA, 93% with a murine B lymphocyte cDNA, and 91% with a rat basophilic leukemia cell cDNA. In contrast to reports on the activities of nonhuman forms of P58, the purified expressed human P58 showed no carnitine acyltransferase or protease activities. However, it did have protein disulfide isomerase activity, indicating that the physiological activity of human liver P58 may be attributed, at least in part, to this activity.