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W Tanner

Publications and source records attributed to W Tanner.

At least 91 records · Page 5Linked to original sources
PubMed1979

The effect of intracellular pH on the rate of hexose uptake in Chlorella.

The rate of hexose uptake by Chlorella is reduced by uncouplers such as carbonyl cyanide p-trifluoromethoxyphenyl hydrazone or dinitrophenol even before concentration equilibrium is reached. The addition of uncouplers changes the membrane potential and the intracellular pH. The membrane potential does not influence the initial velocity of net sugar uptake, whereas manipulation of the cell pH by means of dimethyloxazolidinedione or by butyric acid uncovered a dramatic influence of cell pH on the rate of hexose uptake: at pH values of 7.5--6.8 maximal rate of uptake is observed but at more acid pH a strong inhibition takes place with virtually total blockage of uptake at pH 6.1. The decrease of cell pH to 6.1 in the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone could therefore account for the decrease in hexose transport rate. It was shown that the intracellular pH as such determines the rate of uptake and not the pH difference between inside and outside; the transport rate did not correlate with delta pH.

Biological Transport↗
PubMed1979

Greatly decreased susceptibility of nonmetabolizing cells towards detergents.

The addition of different detergents to Chlorella cells that had previously accumulated 6-deoxyglucose causes a rapid release of the hexose analogue into the medium. This effect is independent of the nature of the detergent and is observed only when the cells are in an energized state. Thus, in the presence of the uncoupler p-trifluoromethoxycarbonylcyanide phenylhydrazone or of inhibitors such as N-ethylmaleimide, the cells show a greatly reduced susceptibility towards detergents. Similarly, the detergent-induced loss of accumulated alpha-aminoisobutyric acid from Saccharomyces cerevisiae and of potassium from Escherichia coli is also strongly affected by the energy state of the cells. The differential susceptibility of energized and nonenergized cells was observed at all detergent concentrations tested. Measurements of substrate efflux at different concentrations of Triton indicated that only Triton monomers are responsible for the increase in permeability. The absorption of [14C]Triton X-100 by Chlorella and the binding of detergent to the cells were measured in the presence of metabolic inhibitors. Again, nonenergized cells bound a significantly lower amount of Triton X-100. The amphiphilic antibiotic nystatin produced effects on cell permeability similar to those of detergents, whereas toluene, which is apolar, gave opposite results.

Aminoisobutyric Acids↗
PubMed1979

Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm.

In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [(14)C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [(14)C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.All of these reactions occur with the highest specific as well as total activity in the endoplasmic reticulum fraction. Whereas a membrane fraction lighter than the endoplasmic reticulum also contains a significant amount of glycosyl transfer activity, the enzymes seem to be present in very low amounts in glyoxysomes and mitochondria. The activities of the various reactions increase severalfold during the first days of germination, i.e. the time of rapid formation of glyoxysomes. The importance of these results in relation to intracellular protein segregation is discussed.

Journal Article↗
PubMed1979

Localization of dolichyl phosphate- and pyrophosphate-dependent glycosyl transfer reactions in Saccharomyces cerevisiae.

Membranes from Saccharomyces cerevisiae protoplasts were fractionated on a continuous sucrose gradient. Six bands were obtained, which contained altogether about 15% of the total cell protein. From their densitites, their behavior in the presence and absence of Mg2+ ions, and the distribution of marker enzymes, it was possible to identify fractions enriched in rough and smooth endoplasmic reticulum and in mitochondria. All glycosyl transfer reactions investigated where dolichyl phosphates served as glycosyl acceptors or where dolichyl phosphate- and pyrophosphate-activated sugars served as glycosyl donors showed the highest specific activity and up to 75% of the total activity in the endoplasmic reticulum. This was the case for the reactions involved in the formation of O-glycosidic as well as N-glycosidic linkages in yeast glycoprotein biosynthesis. Membrane fractions enriched in plasmalemma contained less than 3% of the corresponding activities.

Acetylglucosamine↗
PubMed1978

Biosynthesis and characterization of large dolichyl diphosphate-linked oligosaccharides in Saccharomyces cerevisiae.

Yeast membranes incorporate radioactivity from GDP[14C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components. The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditions. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol. When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharides is formed besides the previously-described Dol-PP-(GlcNAc)2 mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [14C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.

Cell Membrane↗
PubMed1978

Essential Sulfhydryl Group in the Transport-catalyzing Protein of the Hexose-Proton Cotransport System of Chlorella.

The polyene antibiotic nystatin transforms the sugar-proton contransport system of Chlorella to a mere facilitated diffusion system. This experimental condition was used to test the sugar-translocating unit of the active uptake system for possible essential sulfhydryl groups. It could be shown that the catalyzed translocation of sugar is sensitive to the sulfhydryl-reactive compound N-ethylmaleimide. Sugar flow by passive leak as induced by the detergent Triton X-100 is not affected by sulfhydryl reagents. These results show that the sugar-translocating carrier protein possesses a sulfhydryl group, which is essential for its function.

Journal Article↗
PubMed1977

The formation of glycosidic bonds in yeast glycoproteins. Intracellular localisation of the reactions.

Membranes of Saccharomyces cerevisiae were separated on urografin gradients. The specific activity of the light membranes (endoplasmic reticulum), the Golgi-like vesicles and the plasma membrane in transferring mannosyl residues from GDP-mannose to mannoproteins and to dolichyl monophosphate has been determined. The first mannose of the O-glycosidically linked manno-oligosaccharides is incorporated with the highest specific activity by the endoplasmic reticulum. The incorporation of the second to fourth mannosyl groups is catalysed with increasing activity also by the Golgi-like vesicles and the plasma membrane. The incorporation of mannosyl groups into weak alkali-stable positions (N-glycosidically linked chains) is carried out with almost the same specific activity by all three membrane fractions, however, dolichol-dependent and -independent steps could not be distinguished as yet. The results are discussed in terms of a sequential addition of sugar residues along the route of export of the mannoprotiens. The dolichol-dependent steps seem to occur on the endoplasmic reticulum and thus very early in the event.

Cell Membrane↗
PubMed1977

Characterization and partial purification of an inducible protein related to hexose proton cotransport of Chlorella vulgaris.

1. Cells of Chlorella vulgaris induced for hexose transport contain a membrane-bound protein component that is missing in non-induced cells. This was shown by double labelling experiments with [14C]phenylalanine and [3H]phenylalanine applying the method of Kolber and Stein [Nature (Lond) 209 (1966) 691]. The specific protein is completely absent from soluble fractions. 2. An enrichment of the double-labelled peak was observed in membrane fractions enriched in plasmalemma. The best purification was obtained when cell walls were purified; the residual membranes attached to the walls contained the transport protein with a 12-times-higher specific activity than the crude extract. 3. The transport protein had the characteristics of an intrinsic membrane protein. Its molecular weight was found to be 30 000 on sodium dodecylsulphate gels. The protein did not show sugar binding activity, however. 4. Induced cells lose their state of induction with a half life of about 4 h; 7 h after induction the double-labelled transport protein is no longer detectable.

Biological Transport, Active↗
PubMed1976

The determination of the membrane ptoential of Chlorella vulgaris. Evidence for electrogenic sugar transport.

From data on the accumulation of tetraphenylphosphonium within Chlorella vulgaris cells, it can be estimated that these cells possess a membrane potential of --120 to --150 mV (inside negative). Under anaerobic conditions as well as in the presence of uncoupling agents the membrane potential drops to about -60 to -80 mV. Nystatin (50 mug/ml) abolishes it almost completely. Since it took more than 1 h before the tetraphenylphosphonium equilibrium was reached, this method could not be used to measure relatively fast transient changes in membrane potential. However, the rate of influx of tetraphenylphosphonium is also directly dependent on membrane potential and can be followed within minutes. Using this phenomenon as an indicator for membrane potential a brief transient depolarisation was detected after the addition of sugars taken up by Chlorella via the proton cotransport system. The depolarisation was absent from cells not induced for sugar uptake and induced cells did not show it with substances not transported, like mannitol. The maximal depolarisation observed amounted to about 70 mV; after 1 min, however, the membrane potential returned to a value about 25 mV less negative than the one before sugars was added. The results demonstrate that sugar uptake in Chlorella is electrogenic. The delta pH plus membrane potential measured for Chlorella completely cover the energy required to explain the 1600-fold accumulation of 6-deoxyglucose experimentally observed.

Anaerobiosis↗