Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae.

Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography. The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities. The native protein consists of four subunits with a approximately 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3. The equilibrium constant for fumarate hydration is about 4.3 (25 degrees C, pH 7.5), the Michaelis constants for fumarate and 1-malate are approximately 30 microM and approximately 250 microM, respectively. The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type. The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method. The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 A, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis.     

A nystatin-resistant mutant of Saccharomyces cerevisiae: isolation and characterization by electron microscopy and chemical analysis of whole cells, cell-walls and protoplasts

A mutant of Saccharomyces cerevisiae NCYC 239 with a high minimum inhibitory concentration (35 micrograms ml-1) for nystatin, compared to that of the parent strain (2 micrograms ml-1), was derived by a series of subcultures in media containing increasing antibiotic concentrations. In the absence of nystatin, the growth rate of the mutant was significantly lower than the parent strain, although mean cell-size and size-distribution were similar. No differences between strains were detectable by electron microscopy. Analysis of whole cells showed the total sterol present and the ratio of ergosterol:24(28)dehydroergosterol was similar. However, there were marked differences in amino acid content and chain-length of fatty acids in the cell wall, and protoplasts from resistant cells had decreased amounts of unsaturated fatty acids. It is suggested that alterations in cell wall components in the mutant may be directly linked to the mechanism of nystatin resistance.     

On the nature of dominance in the ad2 locus of Saccharomyces cerevisiae

Summary A negative complementation experiment was used to study dominance in the ad₂ gene of Saccharomyces cerevisiae. The wild type allele showed near complete dominance over both the remedial and the inactive mutant alleles. The remedial allele was dominant to a lesser extent (in 77.4% of the combinations) over the inactive mutant allele. In 22.6% of the cases, the inactive allele was able, contrary to expectation, to dominate and impose its inactivity on its remedial partner (negative complementation).At the protein level, these results suggest that the wild type protein, which is the outcome of long evolutionary selection, has the most stabilized activity. The remedial protein, although superior to that of the completely inactive mutant, was not stable enough to always resist the inactivating influence of its defect partner.Part of a thesis of the Faculty of Mathematical and Natural Sciences of Freiburg University.     

ESR study of copper(II) retention by entire cell, cells walls, and protoplasts of Saccharomyces cerevisiae

Copper retention by whole cells, protoplasts, and isolated cell walls of Saccharomyces cerevisiae was investigated in the absence of any energy source in the medium. The cell walls accounted only for a small fraction of the cation retention by whole cells. ESR results showed that copper was not bound only at the outer face of the plasma membrane, but it was also distributed in the plasma membrane and (or) in the cytoplasm. ESR studies also showed that, in all three systems, copper was chelated by peptides or proteins. The binding sites were formed by an amide and a strongly complexing ligand such as an amine. Their configuration depended upon pH: in slightly acidic conditions, copper was bound by the oxygen of the amide; at basic pH, NHCO became deprotonated and the negatively charged nitrogen bound to the metal.     

Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.     

Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.     

Induction of the cytoplasmic 'petite' mutation by chemical and physical agents in Saccharomyces cerevisiae.

A range of physical and chemical agents induce the mitochondrial 'petite' mutation in the yeast Saccharomyces cerevisiae. DNA intercalating agents as well as chemicals which can interfere with DNA synthesis induce this mutation, but only in growing cells. Many chemical or physical agents that produce a DNA lesion which is not simply reversed can induce various levels of the petite mutation, and may be more effective in non-growing cells. A limited number of chemicals act like ethidium bromide, inducing a high frequency of petites which is partially reversible with increasing concentration or time. The ability of a specific compound to be transported into mitochondria or its affinity for AT base pairs in DNA may determine whether it acts primarily as a nuclear or mitochondrial mutagen. In mammalian cells, some neoplastic changes occur at the mitochondrial level. Analogies between yeast and mammalian mitochondria suggest that agents which increase petite mutagenesis in yeast may have some carcinogenic potential. Although some types of petite inducer may have potential as antitumour drugs, those which are very effective antimitochondrial agents appear to be too toxic for therapeutic use. A process comparable to early stages in petite mutagensis occurs in human degenerative diseases and it seems possible that a consequence of exposure to petite mutagens could be an increase in the rate of degenerative diseases or of the aging process.     

Production and reutilization of an extracellular phosphatidylinositol catabolite, glycerophosphoinositol, by Saccharomyces cerevisiae.

Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns). We now report that S. cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability. A wild-type strain uniformly prelabeled with [3H] inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol. This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization. In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered. An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho [3H]inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2. Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporter-independent mechanism. The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent. Labeling studies employing [14C]glycerophospho[3H] inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.     

Crystallization and preliminary X-ray diffraction study of the heat-labile enterotoxin B subunit produced by enterotoxigenic Escherichia coli

Heat-labile enterotoxin LT produced by enterotoxigenic Escherichia coli is composed of A and B subunits. The A subunit is enzymatically active; whereas, through the action of the B subunit, the toxin binds to the receptor, a GM1 ganglioside present on the cell surface. Crystals of the LT-B subunit were formed at room temperature by vapor diffusion with polyethylene glycol in the presence of the non-ionic detergent beta-octylglucoside. The crystals were characterized by X-radiation as orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions of a = 224.1 A, b = 65.3 A, c = 118.4 A. They diffract X-rays to a resolution of at least 2.5 A and are stable to X-rays.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

Kinetics of β-glucan and chitin formation by cells and protoplasts of the yeastSaccharomyces cerevisiae

Intact cells and protoplasts of the yeastSaccharomyces cerevisiae were grown in liquid medium with radioactive glucose as the sole carbon source, and the kinetics of radioactivity incorporation into β-glucan and chitin fractions were measured and compared. While the synthesis of β-glucan by protoplasts started early after their being suspended in the growth medium, the onset of chitin formation was delayed about 3 h and, unlike β-glucan, its formation depended on synthesis of undisturbed protein. In the intact cells, the ratio of β-glucan to chitin was constantly around 12 during growth; while in protoplasts this ratio steadily decreased in the course of cultivation and reached the value of 1.1 after 16h, which can be ascribed to the higher rate of chitin formation by protoplasts in comparison with normal cells. The deproteinized polysaccharide nets formed on the surface of protoplasts that had been incubated in the presence and in the absence of cycloheximide did not differ substantially in their morphology. The only observed difference was the presence of granular material in the samples from control protoplasts grown in the absence of cycloheximide.     

Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production.     

Role of intestinal mucus in crystal biogenesis: an electron-microscopical,diffraction and X-ray microanalytical study

Summary In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needleshaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.     

The effect of liquid holding on chemical induced lethality and mitotic gene conversion in Saccharomyces cerevisiae

Summary Mitotic gene conversion was induced with a variety of chemical mutagens in a double heteroallelic strain of Saccharomyces cerevisiae. Cells were treated with various mutagens and plated immediately onto selective and nonselective growth medium or else they were subject before plating to liquid holding in buffer for various lengths of time. In respiratory competent cells liquid holding caused a decrease in lethality and in conversion frequencies. Respiratory deficient cells, unable to use a non-fermentable substrate as an energy source, behaved different. Untreated cells started to die in buffer after two days of storage, and moreover, there was a considerable increase in potential convertants i.e. cells giving rise to gene convertants when plated on selective growth media. Respiratory deficient cells treated with various chemical mutagens were still more sensitive to liquid holding. After low, sublethal doses cells started to die after one day of liquid holding already and when plated on media selective for convertants, showed an increasing frequency of gene convertants. Addition of very low concentrations of glucose to the liquid holding buffer post-poned the lethal and convertogenic effects. Higher concentrations of glucose completely abolished sensitivity to liquid holding-induced lethality and genetic alterations. The results are interpreted to mean that in respiratory deficient cells no repair activities are possible to an accumulation of spontaneous lethal damage and genetic alterations which are expressed as gene conversion when an energy source becomes available. Such a repairless condition causes an increased sensitivity to genetically active agents, and provides a useful system to detect genetic effects of slowly reacting agents.     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.     

Oxygen requirements for formation and activity of the squalene epoxidase in Saccharomyces cerevisiae.

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the "mitochondrial" fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the "microsomal" fraction. At 0.03% (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent Km for oxygen of 0.38% (vol/vol) was determined from a crude particulate preparation for the epoxidase.     

Osmotic adjustment and the accumulation of organic solutes in whole cells and protoplasts of Saccharomyces cerevisiae

In the presence of a suitable carbon source, whole cells and protoplasts of Saccharomyces cerevisiae synthesized glycerol as a compatible organic solute in response to increased external osmotic pressure. Boyle-van't Hoff plots showed that protoplasts, and non-turgid cells, exhibited a linear relationship between volume and the external osmotic pressure (i.e. they behaved as near-ideal osmometers), and that both protoplasts and cells have a component which is not osmotically responsive--the non-osmotic volume (NOV). Glycerol levels in whole cells and protoplasts were elevated by increased external osmotic pressure over a similar time-scale to the period of exponential cell growth, reaching a maximum value at 6-12 h and declining thereafter. This suggests that the restoration of turgor pressure in whole cells was not the sole regulator of glycerol accumulation. Stationary phase whole cells had negligible levels of intracellular glycerol after growth in a medium of raised osmotic pressure. However, intracellular trehalose synthesis in these cells began earlier and reached a higher maximum level than in basal medium. Once exponential growth had stopped, cell turgor and internal osmotic pressure decreased somewhat. These new, lower values may be determined by the extent of trehalose accumulation in stationary phase cells.