Chapter 8 Analytical Methods for Yeasts

Stewart, P. R.

doi:10.1016/s0091-679x(08)60955-3

Cited by 91 publications

(48 citation statements)

References 57 publications

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“…As noted above, this was equivalent to 15 nMCu. Total cell protein was determined on all cell samples analysed for copper accumulation (Stewart, 1975), whereas protein in soluble extracts was determined by BCA (bicinchoninic acid) assay (Pierce) using bovine serum albumin as standard (Smith et al, 1985). A crude sulphite reductase fraction was isolated and assayed as described by Yoshimoto & Sat0 (1968).…”

Section: Methodsmentioning

confidence: 99%

Distribution of 64Cu in Saccharomyces cerevisiae: cellular locale and metabolism

Lin

Crawford²,

Kosman³

1993

Journal of General Microbiology

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The metabolism of copper in the yeast Saccharomyces cerevisiae has been studied with respect to the distribution and stability to exchange of newly arrived @Cu. Cells pre-incubated with 10 ~M -C U~+ accumulated @Cu into two pools distinguishable by cellular locale and lability to exchange with extracellular cold copper. One pool was nonexchangeable and was localized to protoplasts. Size-exclusion chromatography of a soluble cell (protoplast) extract showed that this 64Cu was associated with up to four species. Two were identified as copper metallothionein and Cu,Zn superoxide dismutase based on comparisons of chromatograms derived from strains in which the genes for these two proteins had been deleted. A third species was identified as copper-glutathione based on chromatographic and biochemical assays. A second pool was exchangeable and was localized to the cell wall. In contrast to its rapid copper-stimulated exchange (t; M 1 min), this pool exhibited only slow efflux (10% 64Cu loss per 60 min). ZnZ+ did not stimulate the loss of 64Cu from this pool indicating that it was selective for copper, This pool was released into the supernatant upon protoplast formation and was found in the cell wall debris obtained when cells were mechanically disrupted. This 64Cu eluted in the void volume (peak P,) of the column used to sizefractionate copper-binding species. The metal in P, was exchangeable in vivo and in vitvo. However, the corresponding chromatographic fraction obtained from copper-naive cells when labelled in vitro could bind less than 20% of the @Cu bound to it in vivo indicating that the deposition of copper in this pool was primarily celldependent. In fact, this deposition was shown to be dependent on the cellular reduction of medium sulphate or sulphite to the level of sulphide, or on the addition of sulphide to the 64Cu uptake buffer. 64Cu in the nonexchangeable protoplast pool was not mobilized by cellular sulphide generation, indicating that cellular sulphide generation did not causally lead to the partitioning of @Cu to the cell wall pool. The data indicate that the appearance of copper sulphide(s) on the cell wall in S. cerevisiae is gratuitous and does not represent a sulphidebased mechanism of copper resistance in this yeast.

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Section: Methodsmentioning

confidence: 99%

Distribution of 64Cu in Saccharomyces cerevisiae: cellular locale and metabolism

Lin

Crawford²,

Kosman³

1993

Journal of General Microbiology

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“…The cell wall preparation was extracted with 0.25 M-Na,C03 (500 ml per g dry wt) at 90 "C for 45 min. The soluble extract of alkali-soluble glucan and mannoprotein (Stewart, 1975) was discarded and the insoluble fraction was washed extensively with distilled water. The insoluble residue was treated with pancreatin as described by Ram et af.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Wall Glucans from Yeast, Hyphal and Germ-tube Forming Cells of Candida albicans

1984

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Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germtube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained fl( 1+3)-and p( 1+6)-chains but no mixed intra-chain p( 1+3)/( 1+6) linkages. Methylation and 3C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (674% to 76.6%) of /3( l-+6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29.6% to 38.9% fl( 143) and 43-3 % to 53-2 % fl( 146) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67.0% fl(1-3) and 14% fl(1+6) linkages. Branch points accounted for 6-77;, 12.3% and 17.4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively. I N T R O D U C T I O NThe cell wall of the dimorphic yeast Candida albicans is qualitatively similar in composition to that of Saccharomyces cerevisiae (for reviews see Cabib et al., 1982; Ballou, 1982;Duffus et al., 1982). Glucans, which include the main structural component, and mannoproteins constitute at least 80% of the C. albicans wall (Sullivan et al., 1983). Chitin, 0.6% of C. albicans yeast walls (Sullivan et al., 1983), is located mainly (90%) in the bud scars but is also dispersed around the wall (Cabib et al., 1982;Tronchin et al., 1981).Germ-tube forming cells, which constitute the initial stage in the yeast-mycelium transition, and hyphal cells contain three to five times more chitin than yeast cells (Braun & Calderone, 1978;Sullivan et al., 1983;Chattaway et al., 1968). Cassone et al. (1973) and Scherwitz et al.( 1 978) have shown that the layered organization of the wall, visualized by electron microscopy, changes significantly with the onset of germ-tube formation; in particular an inner electrontranslucent layer becomes dominant in the germ-tube.Previous studies suggest that the fine structure of the cell wall glucan changes with the age of the cells and the morphology. Gale et al. (1980) showed that the increased resistance to amphotericin exhibited by stationary-phase yeast cells was due to changes in the cell wall and that treatment with glucanases decreased the resistance. Chattaway et al. (1968) reported that although the alkali-insoluble glucan fractions from hyphal and yeast cells had similar glucose contents, treatment with a fl-glucanase released 19% and 39% of the glucose, respectively.Reports of detailed structural analysis of the C. albicans glucans are limited. Bishop et al. (1960) and Yu et al. (1967) extracted a glucan fraction from C. albicans serotypes A and B with boiling 3 % NaOH. Non-reducing terminal and fl(l+6)-linked residues accounted for more than 70% of the glucose. These preparations contained only a trace (serotype A) and 177; (serotype B) of p( 1+3)-linked residues. However, the alkali-soluble fraction represents less ...

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“…Yeast samples (40 mg dry wt) were washed three times with ice-cold water and stored at -17 "C prior to analysis. Carbohydrates were fractionated according to the method of Trevelyan & Harrison (1956) as reported by Stewart (1975), and fractions were analysed in triplicate using the anthrone procedure (Stewart, 1975). Glucose and dry weight were determined as described previously (Quain & Haslam, 1979).…”

Section: Methodsmentioning

confidence: 99%

Changes in Glucose 6-Phosphate and Storage Carbohydrates during Catabolite Derepression in Saccharomyces cerevisiae

Quain¹,

Haslam²

1979

Journal of General Microbiology

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Changes in the cellular content of glucose 6-phosphate and storage carbohydrates during cataboli te derepression were measured during the diauxic growth of Saccharornyces cerevisiae on glucose (1 %, w/v) medium. After the exhaustion of glucose there were transient increases in glucose 6-phosphate, alkali-soluble glycogen and trehalose, reflecting enhanced gluconeogenesis, followed by an increase in acid-soluble glycogen. During the subsequent adaptation to growth on ethanol the amounts of glucose 6-phosphate, trehalose and glycogen returned to basal levels. Thus storage carbohydrates, of which glycogen is quantitatively the most important, appear to act as the energy source for catabolite derepression.

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Chapter 8 Analytical Methods for Yeasts

Cited by 91 publications

References 57 publications

Distribution of 64Cu in Saccharomyces cerevisiae: cellular locale and metabolism

Distribution of 64Cu in Saccharomyces cerevisiae: cellular locale and metabolism

Analysis of Wall Glucans from Yeast, Hyphal and Germ-tube Forming Cells of Candida albicans

Changes in Glucose 6-Phosphate and Storage Carbohydrates during Catabolite Derepression in Saccharomyces cerevisiae

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