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PubMedDate not supplied

Lack of correlation between trehalase activation and trehalose-6 phosphate synthase deactivation in cAMP-altered mutants of Saccharomyces cerevisiae.

The rise in cAMP level that follows the addition of glucose or 2,4-dinitrophenol (DNP) to stationary-phase cells of Saccharomyces cerevisiae was accompanied by a marked activation of trehalase (3-fold increase) and a concomitant deactivation of trehalose-6 phosphate synthase (50% of the basal levels). In glucose-grown exponential cells, which are deficient in glucose-induced cAMP signalling, the addition of glucose also prompted a decrease in trehalose-6 phosphate synthase, but had no effect on trehalase activity. Mutants defective in the RAS-adenylate cyclase pathway (ras1 ras2 bcy1 strain), as well as mutants containing greatly reduced protein kinase activity either cAMP-dependent (tpkw1 BCY1 strains) or cAMP-independent (tpk1w1 bcy1 strains), were unable to show glucose- or DNP-induced trehalase activation but still displayed a clear decrease in trehalose-6 phosphate synthase activity upon addition of these compounds. These data suggest that the activity of trehalose-6 phosphate synthase, as opposed to that of trehalase, is not controlled by the cAMP signalling pathway "in vivo". Trehalose-6 phosphate synthase was competitively inhibited by glucose (Ki = 15 mM) and resulted unaffected by ATP in assays performed "in vitro".

Cyclic AMP↗
PubMed1996

Regulation of genes encoding subunits of the trehalose synthase complex in Saccharomyces cerevisiae: novel variations of STRE-mediated transcription control?

Saccharomyces cerevisiae cells show under suboptimal growth conditions a complex response that leads to the acquisition of tolerance to different types of environmental stress. This response is characterised by enhanced expression of a number of genes which contain so-called stress-responsive elements (STREs) in their promoters. In addition, the cells accumulate under suboptimal conditions the putative stress protectant trehalose. In this work, we have examined the expression of four genes encoding subunits of the trehalose synthase complex, GGS1/TPS1, TPS2, TPS3 and TSL1. We show that expression of these genes is coregulated under stress conditions. Like for many other genes containing STREs, expression of the trehalose synthase genes is also induced by heat and osmotic stress and by nutrient starvation, and negatively regulated by the Ras-cAMP pathway. However, during fermentative growth only TSL1 shows an expression pattern like that of the STRE-controlled genes CTT1 and SSA3, while expression of the three other trehalose synthase genes is only transiently down-regulated. This difference in expression might be related to the known requirement of trehalose biosynthesis for the control of yeast glycolysis and hence for fermentative growth. We conclude that the mere presence in the promoter of (an) active STRE(s) does not necessarily imply complete coregulation of expression. Additional mechanisms appear to fine tune the activity of STREs in order to adapt the expression of the downstream genes to specific requirements.

Blotting, Northern↗
PubMed1993

A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant.

A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced. The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa. The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase. Complementation studies with E. coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent. Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1. A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified.

Amino Acid Sequence↗
PubMed1995

Production of trehalose phosphorylase by Catellatospora ferruginea.

A range of microorganisms was screened for new and high producer strains of trehalose phosphorylase (EC 2.4.1.64). Trehalose phosphorylase activity was found in cells of actinomyctes of the genera Actinomadura, Amycolata, Catellatospora, Kineosporia, and Nocardia. Among them, Catellatospora ferruginea showed the highest enzyme activity. Trehalose phosphorylase from C. ferruginea was able to catalyse both the phosphorolysis of trehalose into beta-glucose 1-phosphate and D-glucose and the synthesis of trehalose from beta-glucose 1-phosphate and D-glucose.

Actinomycetaceae↗
PubMed2005

Isolation of trehalose-6-phosphate phosphatase gene from tobacco and its functional analysis in yeast cells.

Trehalose is a nonreducing disaccharide composed of two glucose units joined by an alpha, alpha-1, 1 linkage, and has been found in bacteria, yeast, fungi, invertebrates and plants. Accumulation of trehalose in organisms plays a role in enhancing the stress tolerance. A trehalose-6-phosphate phosphatase gene, NtTPPL, was isolated from tobacco in this report. The predicted NtTPPL protein has a putative trehalose_PPase domain. The transcription of the NtTPPL gene was significantly induced by heat stress, and was only slightly induced by NaCl, PEG and low-temperature treatments. When expressing in yeast tps2 mutant, NtTPPL rescued the mutant phenotype under high temperature. This result indicated that NtTPPL functioned as a trehalose-6-phosphate phosphatase in yeast and may play similar roles in plants.

Amino Acid Sequence↗
PubMed2002

Sensitivities to juvenile hormone and ecdysteroid in the diapause larvae of Omphisa fuscidentalis based on the hemolymph trehalose dynamics index.

The final instar larva of the bamboo borer, Omphisa fuscidentalis, is in diapause for 9 months from September to the following June. Trehalose and ecdysteroid concentrations in hemolymph were measured through the larval diapause period and in the pupal stage. The ecdysteroid concentration remained low until November, followed by a gradual increase to about 30 ng/ml in May. The trehalose concentration remained at levels ranging between 40-50 mM until May, and decreased to an almost undetectable level after pupation. Since a juvenile hormone analogue (JHA), methoprene, is capable of terminating diapause by stimulating larval prothoracic glands, we examined its effects on ecdysteroid and trehalose concentrations in larvae in December and February. The hemolymph ecdysteroid increased more quickly in February than in December, indicating that the sensitivity of the prothoracic glands to JHA increased towards the end of diapause termination. Similarly, hemolymph trehalose in February decreased within a few days after JHA application, while in December the decrease occurred in the third week. Exogenous 20-hydroxyecdysone (20E) caused a decrease in trehalose concentration in a dose-dependent manner. The effective dose of 20E, however, did not change from January until April, implying that the sensitivity of tissue(s) to 20E may not change until the end of diapause. Taken together, our results suggest that the sensitivities of tissues to JH and 20E do not increase simultaneously with the progress of diapause development and that termination of larval diapause is not associated simply with the restoration of hormone deficiencies.

Journal Article↗
PubMed2001

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune.

Initial-velocity measurements for the phospholysis and synthesis of alpha,alpha-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before alpha,alpha-trehalose, and alpha-D-glucose is released before alpha-D-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to D-glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of D-glucal, a competitive dead-end inhibitor against D-glucose (K(i)=0.3 mM at pH 6.6 and 30 degrees C), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2--7.8 and 5.5--6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of k(cat)/K are interpreted in terms of monoanionic phosphate and alpha-D-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The K(i) value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of D-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and alpha-D-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg(2+) that is non-dissociable in the presence of metal chelators. Measurements of the (26)Mg(2+)/(24)Mg(2+) ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi.

Amino Acid Sequence↗
PubMed1992

Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation.

Trehalose-6-phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose-6-phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose-6-phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose-6-phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.

Amino Acid Sequence↗
PubMed2000

Synthesis, accumulation and hydrolysis of trehalose during growth of peanut rhizobia in hyperosmotic media.

We examined and compared the activities of synthetic and hydrolytic enzymes involved in trehalose metabolism, in three peanut rhizobia strains grown in control, hypersaline, and non-ionic hyperosmotic media. Results indicated that the effects of hyperosmolarity on the synthesis and the degradation of the disaccharide were diverse. In the salt-tolerant slow-growing strain Bradyrhizobium sp. ATCC 10317, we observed increased synthesis and accumulation of trehalose under hyperosmolarity imposed by either NaCl or PEG-8000. In the other two peanut rhizobia strains, the disaccharide level did not change under hypersalinity. In the salt-sensitive slow-growing strain Bradyrhizobium sp. USDA 3187, intracellular trehalose diminished in late stationary phase-cells grown with PEG, this reduction was accompanied by both an increased activity of synthetic enzymes and a decreased activity of trehalase. In the salt-tolerant fast-growing strain Rhizobium sp. TAL 1000, we also observed a reduction of intracellular trehalose under PEG-mediated growth, this decrease was early and transiently accompanied by an enhancement of trehalase activity, afterwards, the activity of synthetic enzymes augmented.

Arachis↗
PubMed1995

Toxicology of cupric salts on honeybees. IV--Gluconate and sulfate action on hemolymph trehalose activity in vivo and in vitro.

A biphasic increase of hemolymph glucose levels was observed following injection to bees of cupric gluconate or sulfate, both potent agents for the control of Varroa jacobsoni, a parasitic mite of hives. The simultaneous injection to bees of 0.3 microM BAYg5421 (an inhibitor of alpha-glucosidases) quenched the response, suggesting a direct effect of 2 nmol/bee cupric ions on trehaloses' activity. One nanomol of injected cupric gluconate increased the trehalose (Tre) activity by 233% in crude hemolymph extracts at 1 mM trehalose concentration, and exhibited biphasic dose-related effects with a maximum 15% increase at 0.5 mM cupric ion and a stabilized 20% inhibition from 4 mM, regardless of the anionic moiety. Upon partial purification of the enzyme complex, two fractions (FI = 75% and FII = 25% of total activity) were isolated that exhibited, respectively, less and more marked positive cooperatively than crude extract. Form I showed almost no susceptibility to either cupric derivatives, which indicated form II as the most likely target, with 68% and 72% increases with 0.25 mM cupric sulfate and 0.5 mM cupric gluconate, in presence of 16 mM trehalose.

Acarbose↗
PubMed1978

ATP, trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum.

Ultra-microfluorometric techniques were adapted to follow several compounds related to energy metabolism through the developmental cycle of Dictyostelium discoideum. Each compound (ATP, trehalose, glucose, and ammonium ion) was found to be present in stalk and/or spore cells. The accumulation of NH4+ was interpreted as an indication of protein degradation, a source of energy in this organism. During the early stages of differentiation NH4+ was localized only in prestalk cells. However, it accumulated in spore cells during culmination such that levels were comparable in the two cell types by the end of development. Trehalose, an energy source for germinating spores, was found in both cell types but was preferentially degraded in stalk cells late in development. Glucose, the degradation product of trehalose, was localized in prestalk cells and varied inversely with trehalose levels. ATP was not localized in a specific cell type during development. However, ATP declined in stalk cells at an earlier stage of development.

Adenosine Triphosphate↗
PubMed2001

Water vapor absorption into amorphous sucrose-poly(vinyl pyrrolidone) and trehalose-poly(vinyl pyrrolidone) mixtures.

Previous studies from this laboratory suggested that a solution model (Flory-Huggins equation) modified by a free volume model (Vrentas equation) could satisfactorily describe water absorption into an amorphous solid composed of a sugar or a polymer. This paper has extended the studies of single solutes to binary mixtures of trehalose-and sucrose-poly(vinyl pyrrolidone) (trehalose-PVP and sucrose-PVP, respectively) either co-lyophilized or individually lyophilized and then physically mixed. Water vapor absorption isotherms of the binary mixtures were determined at 30 degrees C. Co-lyophilized PVP-sugar mixtures take up essentially the same amount of water as predicted by the weight average of individual isotherms, whereas sugar crystallization is significant retarded in the molecular dispersions. The sugar-PVP interaction, as reflected in the Flory-Huggins chi interaction parameter, was estimated by fitting the high relative pressure (p/p(0)) region of the isotherm, at which the system is in a liquid state, with a three-component Flory-Huggins-type model. The estimated sugar-water PVP-water, and sugar-PVP interaction parameters suggest that the solute-water interactions are not significantly affected by the sugar-PVP interaction; that is, the solute-water interaction parameters in a binary solute system are similar to those in the corresponding single solute systems. Based on these interaction parameters, the sucrose-PVP interaction appears to be stronger than that of trehalose-PVP. Manipulation of the interaction parameters suggest that the water vapor absorption isotherm is not a sensitive indicator of possible sugar-PVP interactions. Density, glass transition temperature, T(g), and the heat capacity change, DeltaC(p), at T(g) were determined to estimate the excess water absorption energy due to the plasticizing effect of water using the structural relaxation model, as described by Vrentas. Results suggest that PVP is a better antiplasticizer for sucrose than for trehalose. Consequently, the excess free energy arising from structural relaxation was disproportionally reduced by the presence of PVP in these molecular dispersions. Finally, the entire isotherms of co-lyophilized sugar-PVP mixtures are reasonably described with an extended three-component Flory-Huggins model and Vrentas glass structural relaxation model.

Absorption↗
PubMed1993

A dependable method for the synthesis of [14C]trehalose.

A new method for the preparation of [14C]trehalose was developed, based on the ability of yeast cells to accumulate trehalose under stress. The method is simple and reliable. It utilizes a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted. Thus, exogenously supplied glucose is not metabolized, but is instead converted to trehalose. The [14C]-trehalose obtained is pure, it is hydrolyzed by trehalase, and it is not susceptible to the action of alpha-glucosidase. The yield of this method is in the order of 35% of the [14C]glucose supplied.

Carbon Radioisotopes↗
PubMed1995

Determination of intracellular trehalose and glycogen in Saccharomyces cerevisiae.

A simple, sensitive and nonlaborious enzyme-based method has been developed for determination of both trehalose and glycogen in yeast cells. The method is based on extraction of trehalose and glycogen into a 40 mM acetate buffer (pH 4.8) by mechanical disintegration of the cells in a bead mill. Subsequently, trehalose and glycogen can be hydrolyzed to glucose by the enzymes trehalase and amyloglycosidase, respectively. The formed glucose is quantified by a flow injection analyzer based on the enzyme glucose oxidase. The method gives results comparable to traditional methods but the simplicity of the analysis results in a much lower relative standard deviation. The excellent sensitivity of the glucose analyzer means that as little as 1 microgram trehalose or glycogen can be determined which reduces the required sample volume. This makes the method ideal for physiological studies, e.g., of transients in continuous cultures of Saccharomyces cerevisiae. In addition, a consistent procedure has been derived for pretreatment and storage of samples.

Glycogen↗
PubMed1997

Determination of trehalose by flow injection analysis using immobilized trehalase.

A new method for the determination of trehalose by flow injection analysis (FIA) is described. The basic principle is the hydrolysis of the disaccharide trehalose into its monomer d-glucose by trehalase, a periplasmic enzyme of Escherichia coli. d-glucose is quantified spectrophotometrically after reaction with hexokinase and glucose-6-phosphate dehydrogenase. Trehalase is prepared by osmotic shock from a recombinant E. coli strain and precipitated with ammonium sulfate. The enzyme is immobilized on VA-Epoxy Biosynth from Riedel-de-Haën. The immobilization rate is about 60%. The FIA signals show a nonlinear dependence on the trehalose concentration. The resulting curve corresponds to a second-order polynomial that serves as a calibration function for test samples. Immobilized trehalase was used during a period of 4 months without any loss of suitability. Several samples of fermentation broth were tested. The results are verified by HPLC. Within an interval of 2 to 10 g/L trehalose the recovery is about 100-120% with a precision of 7% (coefficient of variation).

Autoanalysis↗
PubMed1996

Heat shock protein synthesis and trehalose accumulation are not required for induced thermotolerance in depressed Saccharomyces cerevisiae.

Intrinsic and heat shock induced thermotolerance of Saccharomyces cerevisiae was investigated in cells grown on glucose and acetate supplemented media. Heat shocked cells (37 degrees C/30 min), in either medium, exhibited induced synthesis of heat shock proteins (hsp) and trehalose. In all cases, with the notable exception of repressed cells of a relatively thermosensitive strain, heat shock acquisition of thermotolerance also occurred in the absence of protein synthesis and coincident decrease in trehalose accumulation. Results indicted that the marked increase in thermotolerance exhibited by non-fermenting (acetate) cells compared with fermenting (glucose) cells was not closely correlated with levels of hsp or trehalose. It was concluded that mechanisms for intrinsic and induced thermotolerance appear to be different and that growth on acetate endows cells with a biochemical predisposition, other than hsp or trehalose, that confers intrinsic tolerance, a factor which may be subject to heat induced modification.

Acclimatization↗
PubMed1994

Reduction in toxicity for red blood cells in buffered solutions containing high concentrations of 2,3-butanediol by trehalose, sucrose, sorbitol, or mannitol.

Erythrocytes were stored at 4 degrees C in solutions of phosphate-buffered saline containing 2,3-butanediol and 4% (w/w) trehalose, sucrose, sorbitol, or mannitol. The 2,3-butanediol contained 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (2,3-butanediol 97% dl). The concentrations of 2,3-butanediol were 30 and 35% (w/w). A solution of 30% 2,3-butanediol showed relatively low toxicity. Hemolysis was only 2% after 5 h, but increased to 6% after 21 h and reached 60% after 46 h. Adding 4% (w/w) of one of the above compounds drastically decreased the toxicity. The two most efficient were the sugars trehalose and sucrose. With 30% 2,3-butanediol and 4% of any of the four compounds, hemolysis was about 0.6% after 2 days of storage. Furthermore, with trehalose or sucrose, hemolysis remained below 3% for 1 month. With sorbitol or mannitol, hemolysis slowly increased to 2% after 7 days and then increased rapidly. Even with 35% 2,3-butanediol, solutions containing trehalose or sucrose showed low toxicity. Hemolysis was also measured after redilution to buffered solution without 2,3-butanediol and without the additive, to mimic perfusion of organs with cryoprotectants and washing. Minima of hemolysis were observed after a few days of storage. The present solutions also have high glass-forming tendencies. They could be of great interest for organ vitrification.

Blood Preservation↗
PubMed1996

Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose, and polyvinylpyrrolidone.

In the present study, IVF bovine embryos were vitrified using as the cryoprotectants, ethylene glycol plus trehalose plus the polymer, polyvinylpyrrolidone (PVP). In Experiment I, toxicity of the vitrification solution (VS) containing 20% PVP was tested in relation to temperature and exposure time. One hundred percent embryo development was observed with treatment at 5 degrees C for 5 min, whereas only 55.5% embryos were developed when the treatment was carried out at 20 degrees C for 5 min. In Experiment II, embryos were vitrified using one of the three treatments (Treatment A, 40% ethylene glycol (EG); treatment B, 40% EG + 11.3% trehalose; and treatment C, 40% EG + 11.3% trehalose + 20% PVP and rehydrations) was performed directly in mPBS. Highest development (84.1%) and hatching rate (68.2%) were obtained when embryos were vitrified with the vitrification solution used in treatment C. In Experiment III, embryos were vitrified as in Experiment II (treatment C). The development and hatching rates were compared after rehydration in different rehydration solutions. No significant difference was observed among the development and hatching rates when rehydration was carried out in different concentrations of trehalose. Five vitrified-warmed bovine embryos were transferred directly to five recipients and three recipients gave birth to three normal calves.

Animals↗