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Biomedical subjects

W Tanner

Publications and source records attributed to W Tanner.

At least 73 records · Page 4Linked to original sources
PubMed1985

Apparent inhibition of glycoprotein synthesis by S.cerevisiae mating pheromones.

Saccharomyces cerevisiae mating pheromones a and alpha factor strongly inhibit the incorporation of radiolabelled glucosamine into N-glycosylated proteins of corresponding haploid cells. This observation was erroneously interpreted as an inhibition of glycoprotein synthesis. It has turned out that alpha factor causes a 4-5-fold dilution of incorporated [14C]glucosamine with non-radioactive endogenous precursor. In the case of the [14C]chitin synthesized, which does not show inhibition by alpha factor, the lowering of the specific activity of the precursor is exactly compensated for by an increased rate of chitin synthesis caused by alpha factor.

Animals↗
PubMed1985

Selection and characterization of chlorella mutants deficient in amino Acid transport : further evidence for three independent systems.

Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l-arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed.

Journal Article↗
PubMed1984

Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N-glycosylated.

alpha Factor specifically inhibits the synthesis of N-glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in alpha cells or in a/alpha diploids. a Factor has the same effect of alpha cells. The synthesis of O-glycosylated proteins is not inhibited. Although the mating pheromones act like a 'physiological tunicamycin', the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N-glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in alpha-factor-treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by alpha factor; but only in the former case did carbohydrate-free protein accumulate in the cells. alpha Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N-glycosylated proteins are required for the G1/S-phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature-sensitive N-glycosylation mutant is shifted to its restrictive temperature.

Animals↗
PubMed1984

A temperature-sensitive N-glycosylation mutant of S. cerevisiae that behaves like a cell-cycle mutant.

The temperature-sensitive S. cerevisiae mutant alg1-1, defective in the N-glycosylation of proteins, shows a first cycle arrest at the non-permissive temperature of 36 degrees C. The cell number increases by 50% and the absorbance approximately doubles. The budding index of 0.4 at 26 degrees C drops to 0.15 and DNA synthesis quickly comes to a halt at 36 degrees C. When the temperature is lowered again, budding and DNA synthesis start after a lag of 2-3 h; alpha-factor prevents both these processes in cells of mating type a. In addition, cells arrested at 26 degrees C in G1 with alpha-factor also do not start budding at the non-permissive temperature after removal of alpha-factor. The results support recent findings obtained with tunicamycin and suggest that at least one glycoprotein is required for G1-S phase transition in yeast.

DNA, Fungal↗
PubMed1983

Synthesis of retinylphosphate mannose in yeast and its possible involvement in lipid-linked oligosaccharide formation.

A membrane fraction from Saccharomyces cerevisiae as well as a mannosyltransferase purified therefrom was shown to catalyze the transfer of mannose from GDPmannose to retinyl phosphate. The product formed has chromatographic and chemical properties characteristic for retinylphosphate mannose. The enzyme requires divalent cations. Mg2+ is more effective than Mn2+ with an optimum concentration around 25 mM. Amphomycin at a concentration of 0.1 mg/ml inhibits the reaction to 50%. Glycosyl transfer was specific for mannose residues from GDPmannose and did not occur with dolichylphosphate mannose nor with UDP galactose; UDPglucose is a poor donor. Formation of retinylphosphate mannose is inhibited by dolichyl phosphate. This observation as well as similarities between retinylphosphate mannose and dolichylphosphate mannose synthesis in respect to ion requirement, inhibition by amphomycin are suggestive that both reactions are catalyzed by one and the same enzyme. In experiments studying the glycosyl donor specificity in the assembly of lipid-linked oligosaccharide intermediates involved in N-glycosylation of proteins, it could be demonstrated that retinylphosphate mannose can replace dolichylphosphate mannose in the final steps of mannosylation.

Catalysis↗
PubMed1982

Synthesis and possible role of carbohydrate moieties of yeast glycoproteins.

The pathways for protein N- and O-glycosylation in yeast cells are summarized. Evidence is presented that the terminal glucosyl residues of the dolichyl-PP-oligosaccharide intermediate are responsible for decreasing the Km for the peptide to be N-glycosylated. A liposomal model system is introduced that allows the study of a dolichyl phosphate (Dol-P) dependent transmembrane transport of mannosyl residues. The results obtained so far suggest that the mannosylation of Dol-P and the transmembrane translocation of Dol-P-Man are catalysed by the enzyme more or less simultaneously. However, only about 8-10% of the enzyme molecules incorporated into the liposomes seem to carry out the 'coupled' reaction. The glycosylation of carboxypeptidase Y is not required for this protein to reach the vacuole, its target organelle. In the presence of low concentrations of tunicamycin, however, yeast cells do stop growth. This does not seem to be due to the inhibition of secretion of glycoproteins like external invertase. It is postulated that protein glycosylation is crucial for a cell cycle event during the G1 phase.

Asparagine↗
PubMed1982

An obligatory role of protein glycosylation in the life cycle of yeast cells.

In the presence of 2-4 micrograms tunicamycin/ml, yeast cells stop growth after the cell number has increased 1.5-1.7-fold. The cells are arrested in G1: the rate of DNA synthesis greatly decreases and the budding index drops from 0.6 to 0.1 RNA synthesis is inhibited concomitantly, whereas protein synthesis is little affected. It is postulated that for G1/S phase transition to occur one or more proteins have to be glycosylated.

Cell Count↗
PubMed1982

Dolichyl phosphate-mediated mannosyl transfer through liposomal membranes.

A partially purified (up to 1000-fold) mannosyl transferase that catalyzed the reversible reaction GDP-Man + Dol-P in equilibrium Dol-P-Man + GDP was incorporated into liposomes consisting of soybean lecithin and dolichyl phosphate (Dol-P). The enzyme transferred the mannosyl moiety from external GDP-Man to liposome-associated Dol-P. However, when the liposomes were preloaded with GDP, mannosyl residues were also transferred to the inside, giving rise to internal GDP-Man by the reverse reaction. This transfer of an activated sugar through a membrane required the presence of Dol-P and the enzyme in the liposome. Mannosyl residues were not transferred to the inside when the liposomes were preloaded with ADP or GMP. Amphomycin completely inhibited the formation of Dol-P-Man as well as the transfer of mannose into the liposomes. The results are taken as evidence for the open postulated role of dolichols in sugar translocation through membranes. The data are discussed in relation to glycoprotein synthesis at the endoplasmic reticulum.

Biological Transport, Active↗
PubMed1982

Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast.

Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III). The test conditions are described in detail. All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+. The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM. The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1. All reactions were inhibited by nucleoside diphosphates.

Cell Membrane↗
PubMed1981

Glucose induces two amino acid transport systems in Chlorella.

In autotrophically grown Chlorella cells, glucose induces a hexose transport system but, at the same time, the synthesis of two amino acid transport systems is also induced. Thus, the rates of uptake of glycine, L-alanine, L-proline, and L-serine, all of which compete with each other for entry into the cells, increase more than 100-fold when the algae are pretreated with glucose. The rates of L-arginine and L-lysine uptake increase by a factor of 25 to 50. The accumulation of proline and arginine within the cells amounts to 200- and 600-fold, respectively. Glucose does not cause the positive effect on amino acid uptake by serving as metabolic substrate because the nonmetabolizable 6-deoxyglucose also acts as inducer. Cycloheximide prevents the induction. The induced transport system for the four neutral amino acids has a turnover with a half-life of 7 hr, which corresponds closely to the half-life of the hexose transport system. The transport system for the basic amino acids, on the other hand, disappears with a half-life of 25 hr.

Alanine↗
PubMed1981

N-Glycosylation of yeast proteins. Characterization of the solubilized oligosaccharyl transferase.

The enzyme transferring the oligosaccharide from DolPP-(GlcNAc)2(Glc)3 to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from DolPP-[14C]saccharides to the hexapeptide Tyr-Asn-Leu-Thr-Ser-Val. The rate of transfer was linear for 20 min, with about 40% of dolichyl-diphosphate-bound radioactivity transferred to the peptide. The solubilized enzyme has been characterized as follows: 1. The enzyme is most efficiently solubilized (60% of the membrane-associated activity) by 0.5% Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction if present in the test at a concentration of 1%. 2. Divalent cations are absolutely required. 1 mM Mn2+ is optimal; Mg2+ at a concentration of 10mM yields only one third the activity observed with Mn2+. 3. The enzyme transfers besides dolichyl-diphosphate-bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc)2(Man)1 and (GlcNAc)2; the rate decreases in this order. No transfer is observed from DolPP-(GlcNAc)2(Man)9 and from DolPP-GlcNAc. 4. The Km value for DolPP-(GlcNAc)2(Man)9(Glc)3 of 0.5 microM does not differ significantly from that for DolPP-(GlcNAc)2 of 1.2 microM. A broad pH-optimum for the reaction with both substrates was found between 6.5 and 7.7. 5. However, a clear difference in Km values for the hexapeptide was observed with difference dolichol-linked sugar derivatives. With DolPP-(GlcNAc)2 a peptide concentration of 0.6 mM resulted in half-maximal transfer rate, whereas 0.05 mM peptide were sufficient with DolPP-(GlcNAc)2(Man)9(Glc)3 as donor.

Cations, Divalent↗
PubMed1980

Yeast mannosyl transferases requiring dolichyl phosphate and dolichyl phosphate mannose as substrate. Partial purification and characterization of the solubilized enzyme.

The first mannosyl unit of manno-oligosaccharides of fungal mannoproteins is transferred in a dolichyl-phosphate-dependent reaction sequence to serine/threonine residues of the protein. The two membrane-bound enzymes catalyzing this transfer in the yeast Saccharomyces cerevisiae have been solubilized by detergents. The enzyme transferring mannose from guanosine diphosphate mannose to dolichyl phosphate has been purified 18-fold when based on membrane protein and 140-fold when based on total cell protein. The enzyme transferring mannose from dolichyl phosphate mannose to protein has been purified 48-fold and 380-fold, respectively. A HCl-treated cell-wall mannoprotein from yeast served as acceptor protein for the second enzyme. The solubilized enzyme catalyzing the formation of dolichyl diphosphate mannose has a Km for guanosine diphosphate mannose of 7 x 10(-6) M and is saturated with about 0.15 mM yeast dolichyl phosphate. The metal requirement, pH-optima, and the detergent concentration necessary for optimal activity have been determined for both solubilized enzymes.

Chromatography↗
PubMed1979

Effects of gibberellic acid and of tunicamycin on glycosyl-transferase activities and on alpha-amylase secretion in barley.

A crude membrane fraction was prepared from isolated aleurone layers, the secretory tissue of barley grains. The layers were pre-incubated in the presence or absence of the phytohormone gibberellic acid. The membranes catalyzed the transfer of [14C]mannose from GDP-[14C]mannose and of N-[14C]acetylglucosamine from UDP-N-[14C]acetylglucosamine to endogenous and exogenous dolichyl monophosphate. When gibberellic acid was present during the pretreatment the activity of the transferases was increased by a factor of two to three. A significantly increased activity was observable within four hours after the addition of gibberellic acid, whereas the gibberellic-acid-induced secretion of the glycoprotein alpha-amylase started only after 12 h. Tunicamycin inhibited the secretion of alpha-amylase by 60 to 80%. Intracellularly, however, no alpha-amylase was found to accumulate. On the other hand, tunicamycin did not inhibit the rate of total protein synthesis by more than 10%. The possibility is discussed that the synthesis of the protein portion of glycoproteins is specifically inhibited, when glycosylation is prevented.

Amylases↗