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O Touster

Publications and source records attributed to O Touster.

At least 37 records · Page 2Linked to original sources
PubMed1985

Effects of swainsonine on rat liver and kidney: biochemical and morphological studies.

Among the reported effects of the plant toxin swainsonine in animals are a decreased level of Golgi mannosidase II activity, an increase in lysosomal alpha-D-mannosidase activity, oligosaccharide accumulation, vacuolization of cells, and neurological changes. We now find that, in the rat, the alkaloid rapidly induces vacuolization of both liver and kidney cells, but oligosaccharides accumulate only in the latter. We demonstrate by enzyme- and immunocytochemistry that the induced pleomorphic vacuoles are lysosomal in nature. The vacuoles do not appear to be derived from the Golgi apparatus, which retains its typical ultrastructural appearance, but are formed by autophagy. In swainsonine-fed rats, the lysosomal system is highly developed in hepatocytes, Kupffer cells, and cells of the proximal convoluted tubules. The relation of this hypertrophy of the lysosomal system to the known effects of swainsonine on glycoprotein biosynthesis and on Golgi and lysosomal alpha-mannosidases is not clear. In addition, in liver there occurs a marked increase in mitotic figures in the hepatocytes. This occurred in the absence of both cell death and increased liver size as estimated by gross morphology.

Alkaloids↗
PubMed1984

The similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicate that the alkaloid is the principal toxin responsible for the induction of locoism.

A neurological condition resembling that observed in hereditary mannosidosis occurs in animals ingesting spotted locoweed and plants of the genus Swainsona. Swainsonine has been isolated from these plants and has been suggested to be the primary causative agent in inducing the pathological condition. This alkaloid has also been found to increase tissue acid alpha-D-mannosidase levels in rats while lowering liver Golgi mannosidase II levels. In the present study, the effects of locoweed and swainsonine were directly compared for the first time, with the pig as experimental animal. Both increased most lysosomal acid glycosidase activities in most tissues, decreased liver Golgi mannosidase II levels, increased plasma hydrolase levels, and greatly increased tissue oligosaccharide, especially Man5GlcNAc2 and Man4GlcNAc2. These results indicate that swainsonine is the agent in locoweed responsible for the enzymatic and oligosaccharide changes. The behavior of the animals was also similarly affected by swainsonine and locoweed.

Alkaloids↗
PubMed1983

Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal alpha-D-mannosidase, decreases Golgi alpha-D-mannosidase II, and increases the plasma levels of several acid hydrolases.

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D.R.P. Tulsiani, T.M. Harris, and O. Touster, (1982) J. Biol. Chem. 257, 7936-7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal alpha-D-mannosidase and are probably a consequence of abnormal processing of glycoproteins.

Alkaloids↗
PubMed1983

Swainsonine causes the production of hybrid glycoproteins by human skin fibroblasts and rat liver Golgi preparations.

The synthesis of glycoproteins containing N-linked complex oligosaccharides is blocked by swainsonine at the step catalyzed by Golgi mannosidase II (Tulsiani, D. R. P., Harris, T. M., and Touster, O. (1982) J. Biol Chem. 257, 7936-7939). Accordingly, hybrid glycoproteins might be produced in the presence of swainsonine. In this report, we demonstrate that swainsonine causes human skin fibroblasts to synthesize such glycoproteins. In control fibroblasts, there were approximately equal amounts of complex and high mannose glycoproteins. In the presence of swainsonine (10 micrograms/ml), most of the complex glycoproteins were replaced by hybrid types. The principal oligosaccharide had the following structure: (formula; see text) A smaller amount of the asialo hybrid was also produced. The structure of the hybrid was established by Bio-Gel P-4 fractionation of oligosaccharides produced by endoglycosidase H treatment of pronase-derived glycopeptides, followed by examination of the susceptibility of the oligosaccharide to glycohydrolases and by its adsorbability to serotonin-Sepharose 4B. The same hybrid oligosaccharide was produced efficiently by rat liver Golgi membranes in the presence of ([3H] Man)5GlcNAc, UDP-GlcNAc, UDP-Gal, CMP-NeuAc, and swainsonine. Golgi mannosidase II had no action on the hybrid oligosaccharide, and little action on asialo hybrid, but both were converted to the mannosidase II substrate, GlcNAcMan5GlcNAc, by appropriate treatment with neuraminidase and beta-galactosidase. Jack bean alpha-D-mannosidase gave the expected yields of free mannose from the various oligosaccharides studied in this work. Swainsonine should be useful in investigating the role of oligosaccharide structure of glycoproteins because of its ability to alter the oligosaccharide.

Alkaloids↗
PubMed1983

Immunocytochemical localization of alpha-D-mannosidase II in the Golgi apparatus of rat liver.

Mannosidase II is involved in the trimming of alpha-1,6-mannosyl residues during the biosynthesis of glycoproteins containing N-linked oligosaccharides of the complex type. A highly specific polyclonal antibody (IgG) was isolated from rabbits immunized with a homogeneous preparation of mannosidase II prepared from rat liver. With this antibody, light and electron microscopic immunocytochemical studies on rat liver reveal that essentially all mannosidase II in hepatocytes is localized in the Golgi apparatus, the only other site with reaction product being the endoplasmic reticulum. The indirect immunocytochemical method used in this study involved three major steps: exposure of aldehyde-fixed tissue to immune and nonimmune IgG, treatment with staphylococcal protein A labeled with horseradish peroxidase, and incubation in diaminobenzidine to reveal sites of peroxidase activity. The procedures described overcome major problems in immunocytochemistry, allowing preservation of antigenic sites and maintaining adequate ultrastructural integrity. The in situ localization of other carbohydrate-processing enzymes, involved in either trimming or attachment of sugar residues, should be possible with this procedure. Because biosynthetic precursors of the processing enzymes may be revealed by an immunocytochemical approach, it is potentially significant that mannosidase II reaction product is present in areas of the endoplasmic reticulum as well as in the Golgi apparatus.

Animals↗
PubMed1982

Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis.

Rat liver glucosidase II, an endoplasmic reticulum hydrolase involved in the biosynthesis of the N-linked class of glycoproteins, has been purified in good yield to a state approaching homogeneity. The purified enzyme hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, maltose, and the precursor oligosaccharides glucose1-2mannose9N-acetylglucosamine, but it does not act on glucose3mannose9N-acetylglucosamine or p-nitrophenyl-beta-D-glucopyranoside. The ratio of the rate at which glucose is released from p-nitrophenyl-alpha-D-glucopyranoside to that from glucose2mannose9N-acetylglucosamine or glucose1mannose9N-acetylglucosamine remains constant throughout the 8-step purification procedure; thus it appears that a single enzyme is responsible for the activities toward both the artificial and oligosaccharide substrates. The fact that the enzyme cleaves both of the inner 1,3-linked glucosyl residues from the precursor oligosaccharides supports the view that they are linked in the alpha-configuration. The pH dependence of enzymatic activity is quite similar for different substrates, showing a broad optimum between pH 6 and 7.5. Activity toward p-nitrophenyl-alpha-D-glucopyranoside is enhanced by 12 mM 2-deoxy-D-glucose (260-300% activation) and 25 mM mannose (150% activation), but these two compounds inhibit the action of the enzyme toward the precursor oligosaccharides. By isoelectrofocusing the purified enzyme exhibits one form, which has a pI of 3.5-3.8. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates that glucosidase II has a subunit molecular weight of 65,000. Ferguson plot analysis of the behavior of native enzyme in polyacrylamide gels indicates that it is a 262,000-dalton tetramer. Gel filtration gives a molecular weight of 288,000. Several lines of evidence indicate that the enzyme is a glycoprotein.

Animals↗
PubMed1982

alpha-D-Mannosidases of rat liver Golgi membranes. Mannosidase II is the GlcNAcMAN5-cleaving enzyme in glycoprotein biosynthesis and mannosidases Ia and IB are the enzymes converting Man9 precursors to Man5 intermediates.

Current evidence indicates that the trimming of mannosyl residues from intermediates in the biosynthesis of the N-linked oligosaccharides of glycoproteins occurs in the Golgi complex. We now present evidence that mannosidase II (Tulsiani, D. R. P., Opheim, D. J., and Touster, O. (1977) J. Biol Chem. 252, 3227-3233) is the Golgi enzyme that converts GlcNAc Man5 species to GlcNAcMan3 species in completing the mannosyl trimming process required in the biosynthesis of complex type glycoproteins. GlcNAc([3H]Man)5GlcNAc-mannosidase and p-nitrophenyl alpha-D-mannosidase activities copurify throughout the preparative procedure and show the same properties. In addition to mannosidase IA (Tabas, I., and Kornfeld, S. (1979) J. Biol. Chem. 254, 11655-11663), a second alpha-1,2-mannosidase (mannosidase IB) can be prepared from Golgi membranes which is effective in converting Man9GlcNAc to Man5GlcNAc. The two alpha-1,2-mannosidases are very similar in catalytic properties, but they are also distinguishable by several criteria. Although these two enzymes have not been extensively purified, several lines of evidence lead to the tentative conclusion that they are distinct enzymes. They appear to be present in comparable activities in the Golgi membranes and together account for the alpha-1,2-mannosidase activity of these membranes. The particular role of each alpha-1,2-mannosidase remains to be determined.

Animals↗
PubMed1981

Isolation and partial characterization of the oligosaccharide moieties of rat preputial beta-glucuronidase.

The oligosaccharide alditols of rat preputial gland beta-glucuronidase have been isolated by reductive alkaline hydrolysis of the enzyme followed by gel filtration. Since the product was about decasaccharide in size, and as beta-glucuronidase contains about 20 sugar residues per subunit, the enzyme must contain two oligosaccharides per subunit. Two major subfractions were obtained. Chemical and enzymatic studies on the initial oligosaccharide alditol fraction and on the separated subfractions indicated the presence of a mannosyl-chitobiosyl core and permitted formulation of the following structures for the two different oligosaccharide alditols: Man3 leads to alpha [(alpha Man, GlcNAc) leads to Man] leads to beta GlcNAc leads to beta GlcNAc-ol Man 3 leads to 4 leads to alpha[Man2 leads to alpha Man] leads to beta GlcNAc leads to beta GLcNAc-ol.

Animals↗
PubMed1981

Identification of a 2-aminotetrose in a lipid-soluble fraction of rat liver.

Chromatography on DEAE-cellulose of chloroform:methanol:water (10:10:3) extracts obtained from the livers of rats pulsed 50 min with [32P]Pi and D-[1-3H]-mannose yielded two radioactive components which eluted at salt concentrations greater than 0.1 M. One of the components yielded, upon mild acid treatment (20 mM HCl, 100 degrees C, 20 min), a low molecular weight water-soluble component (PO), which contained most of the 32P label associated with the parental lipid-oligosaccharide. PO was periodate-negative, ninhydrin-negative, and permanganate-positive. The phosphate group was stable to strong acid, which converted PO to a single, 32P-labeled product, but the phosphate was readily released by incubation under mild alkaline conditions. Strong alkali produced a product reactive with periodate, ninhydrin, and fluorescamine. When dephosphorylated PO was hydrolyzed with acid and the products were converted to the alditol acetate derivatives, gas chromatography revealed a single major component. Gas chromatography/mass spectroscopy of this compound showed it to be the derivative of a 2-acetamido-2-deoxytetrose. That 2-acetamido-2-deoxytetrose is glycosidically bound in PO is indicated by the fact that PO must be hydrolyzed by strong acid before reduction with NaBD4 is able to incorporate deuterium into the tetrose.

Amino Sugars↗
PubMed1980

In vitro fusion of Acanthamoeba phagolysosomes. III. Evidence that cyclic nucleotides and vacuole subpopulations respectively control the rate and the extent of vacuole fusion in Acanthamoeba homogenates.

Fusion of phagolysosomes has been previously demonstrated to occur during the incubation of phagolysosome-containing homogenates of Acanthamoeba (Oates and Touster, 1978, J. Cell Biol. 79:217-234). Further studies on this system have shown that methylxanthines (0.2 mM) and/or cAMP (0.5-1 mM) markedly accelerate the average rate, but not the extent, of the in vitro phagolysosome fusion process. Adenosine, 5'-AMP, and ADP (0.5-1 mM) were without effect. ATP (0.5-1 mM) caused variable stimulation, whereas beta, gamma-methylene-ATP (1 mM) caused pronounced inhibition, as did GTP (1 mM) and cGMP (1 mM). Stimulation by 3-isobutyl-1-methylxanthine was blocked by GTP, but not by ATP or cAMP. These results indicate that the rate of phagolysosome fusion in Acanthamoeba homogenates may be regulated by cyclic nucleotides, with enhancement of the fusion rate by cAMP and inhibition of the rate by cGMP. The extent of the reaction increased spontaneously and markedly during the first few hours after preparation of the homogenates. This activation appears to be because of a slow conversion of a significant fraction of the vacuole population from a fusion-incompetent to a fusion-competent, cyclic nucleotide-sensitive state.

Adenosine Triphosphate↗
PubMed1979

Demonstration of a rat liver microsomal binding protein specific for beta-glucuronidase.

A binding protein with apparent specificity for beta-glucuronidase has been partially purified from a Triton X-100 extract of rat liver microsomes by affinity chromatography on glucuronidase-Sepharose 2B. It appears that once removed from the membrane, this binding protein self-aggregates to form large macromolecular complexes. With the use of polyacrylamide gel electrophoretic and sucrose density gradient ultracentrifugation assays to monitor the conversion of glucuronidase tetramer to a very high molecular weight complex, it was shown that the binding activity is heatlabile and protease-sensitive. However, binding activity is not influenced by salts, carbohydrates, other proteins or glycoproteins, or by extensive periodate oxidation of beta-glucuronidase, nor does binding occur with any other protein tested. The binding protein does not discriminate against any form of beta-glucuronidase from any rat organ tested. However, the binding protein does show organ localization, being present in the liver and kidney but not the spleen. The possible relationship of this binding protein to egasyn, a membrane protein which stabilizes beta-glucuronidase in mouse liver endoplasmic reticulum, is discussed.

Animals↗
PubMed1979

Studies on dehydrogenases of the glucuronate-xylulose cycle in the livers of diabetic mice and rats.

In view of reports that accessory pathways of glucose oxidation are enhanced in the diabetic state, we have determined the levels of key enzymes of the glucuronate-xylulose cycle in the livers of diabetic mice and rats. Genetically diabetic mice (db/db) were found to have increased levels of two NADP-linked enzymes of this cycle [NADP-xylitol dehydrogenase and NADP-L-hexonate dehydrogenase (aldehyde reductase II)], whereas other NAD- and NADP-linked dehydrogenase activities of the pathway were not changed. On the other hand, the livers of streptozotocin-diabetic mice and rats showed normal levels of all these enzymes. In the course of this study, evidence was obtained for the presence in db/db mouse liver of low molecular weight material inhibitory for glucose 6-phosphate dehydrogenase. The use of these animal models in diabetes research is briefly discussed.

Aging↗
PubMed1978

Lysosomal alpha-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic alpha-D-mannosidases.

Rat liver contains alpha-D-mannosidases in lysosomes, Golgi membranes, and cytosol. The lysosomal enzyme has now been purified approximately 30,000-fold over the crude extract and is free of at least 13 other lysosomal hydrolases. The enzyme has an apparent molecular weight of 335,000 by molecular sieve chromatography and 200,000 by sucrose density centrifugation. It is a glycoprotein, as evidenced by its binding to a concanavalin A affinity column and by a positive periodic acid-Schiff stain. The enzyme has a pH optimum near 4.6. Although it is generally insensitive to a large variety of inorganic salts, chelating agents, and sulfhydryl reagents, prolonged exposure to ethylenediaminetetraacetic acid caused loss of activity, which could be restored by the addition of ZnSO4. Substrate specificity studies were performed on the purified lysosomal alpha-D-mannosidase, as well as on the purified Golgi and cytosolic alpha-D-mannosidases. The three enzymes exhibited only very limited activity on native glycoproteins, but were found to be active on glycopeptides and oligosaccharides, hydrolyzing 1 yields 2 and 1 yields 3 linkages, except that the Golgi enzyme had negligible activity towards the latter linkage. Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three enzymes are not immunologically related. The alpha-D-mannosidase isolated from rat epididymis was found to be immunologically very similar, if not identical, to the lysosomal enzyme isolated from rat liver.

Animals↗