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PubMed1998

Trehalose-mediated protection of the plasma membrane H+-ATPase from Kluyveromyces lactis during freeze-drying and rehydration.

During freeze-drying and rehydration, the activity of the H+-ATPase from the plasma membrane of Kluyveromyces lactis was preserved by increasing concentrations of carbohydrates. When the H+-ATPase was freeze-dried in the absence of carbohydrates the activity was lost. The protective efficiency of carbohydrates was as follows: trehalose > maltose > sucrose > glucose > galactose. Each carbohydrate exhibited the maximal protection at a concentration of 20 mg carbohydrate per milligram of protein or above. No structural changes of the rehydrated H+-ATPase were detected by intrinsic fluorescence measurements. Trehalose, at 20 mg/mg protein, protected the enzyme activity completely during freeze-drying and rehydration. Rehydration temperature was critical; at 20 degrees C or below, activity was fully retained, while at 30, 40, or 50 degrees C activity decreased in proportion with temperature. The trehalose-protected freeze-dried H+-ATPase was stored at different temperatures for up to 60 days. Storage at 4 degrees C resulted in retention of most of the enzymatic activity, while storage at 20 or 30 degrees C resulted in loss of activity. The protection of the H+-ATPase by trehalose suggests that this carbohydrate might protect other membrane enzymes from inactivation during handling.

Cell Membrane↗
PubMed1998

Stabilization of rat liver mitochondrial alanine aminotransferase with ethanol and trehalose.

Rat liver mitochondrial alanine aminotransferase (mALT) is known to be a very unstable enzyme, a property that has hindered efforts to purify it. In this report we examine the possibility of stabilizing mALT with ethanol, trehalose, and protease inhibitors. The presence of ethanol was shown to slow down the inactivation of mALT, increasing its half-life from 1 to 4 h. Trehalose was found to greatly enhance the stability of mALT in a concentration-dependent manner. In the presence of 36.5% trehalose, the half-life of mALT was 85 h. Of the protease inhibitors tested only antipain and chymostatin slowed down the inactivation of mALT but only within the first 24 h following preparation of the crude enzyme. It is concluded that the inclusion of ethanol and trehalose in purification protocols could aid the purification of the enzyme. It is also concluded that the inclusion of protease inhibitors in purification protocols of mALT may not be necessary as its inactivation does not seem to be due to protease activity.

Alanine Transaminase↗
PubMed2001

Beneficial effect of intracellular trehalose on the membrane integrity of dried mammalian cells.

Recently, there has been much interest in using trehalose and other small carbohydrates to preserve mammalian cells in the dried state as an alternative to cryopreservation. Here, we report on the successful preservation of plasma membrane integrity after drying, as a first step toward full preservation of mammalian cells. Trehalose was introduced into cells using a genetically engineered version of alpha-hemolysin, a pore-forming protein; the cells were then dried and stored for weeks at different temperatures with approximately 90% recovery of the intact plasma membrane. We show that protection of the plasma membrane by internal trehalose is dose dependent and estimate the amount of internal trehalose required for adequate protection to be approximately 10(10) molecules/cell. In addition, a minimal amount of water (approximately 15 wt%) appears to be necessary. These results show that a key component of mammalian cells can be preserved in a dried state for weeks under mild conditions (-20 degrees C and 5% relative humidity) and thereby suggest new approaches to preserving mammalian cells.

3T3 Cells↗
PubMed1988

Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations.

The sequence of events following the addition of 0.5 M NaCl to cells of Escherichia coli growing in a minimal mineral medium was investigated. Immediately after upshock the cells took up a large amount of K+ and synthesized approximately half the equivalent amount of glutamate concomitantly. After 30 min the cells started to synthesize trehalose, and after 2 h they had replaced most of their initial osmoprotectants by the carbohydrate. Cell trehalose was rapidly replaced by proline, taken up from the medium when added to the osmoadapting cells. The initial rate of this proline uptake was extremely rapid, and with rates observed of up to 0.6 mmol x min-1 x g-1 of cell protein it was approximately ten times faster than that reported in the literature for non-growing cells. These results indicate that for osmoadaptation of growing cells of E. coli the uptake of proline has priority over the synthesis of trehalose, which in its turn is preferred above K+ and glutamate as osmoprotectants. We observed that two mutants with unknown lesions, but which are known to be impaired in osmoadaptation, were inhibited in replacing K+ and glutamate by trehalose, indicating that this is the basis for their defect in osmoadaptation. Further experiments revealed that neither internal pH nor the membrane potential nor the transmembrane protonmotive force are likely to be involved in osmoadaptation in E. coli. However, during osmoadaptation a high internal potassium concentration appeared to stimulate the derepression of proline-uptake systems (mainly system ProP).

Biological Transport, Active↗
PubMed1982

Localization of trehalase in vacuoles and of trehalose in the cytosol of yeast (Saccharomyces cerevisiae).

Protoplasts of Saccharomyces cerevisiae synthesized and degraded trehalose when they were incubated in a medium containing traces of glucose and acetate. Such protoplasts were gently lyzed by the polybase method and a particulate and soluble fraction was prepared. Trehalose was found in the soluble fraction and the trehalase activity mostly in the particulate fraction which also contained the vacuoles besides other cell organelles. Upon purification of the vacuoles, by density gradient centrifugation, the specific activity of trehalase increased parallel to the specific content of vacuolar markers. This indicates that trehalose is located in the cytosol and trehalase in the vacuole. It is suggested that trehalose, in addition to its role as a reserve may also function as a protective agent to maintain the cytosolic structure under conditions of stress.

Cell Compartmentation↗
PubMed1996

The regulation of trehalose metabolism in insects.

Trehalose is a non-reducing disaccharide comprising two glucose molecules. It is present in high concentration as the main haemolymph (blood) sugar in insects. The synthesis of trehalose in the fat body (an organ analogous in function to a combination of liver and adipose tissue in vertebrates) is stimulated by neuropeptides (hypertrehalosaemic hormones), released from the corpora cardiaca, a neurohaemal organ associated with the brain. The peptides cause a decrease in the content of fructose 2,6-biphosphate in fat body cells. Fructose 2,6-biphosphate, acting synergistically with AMP, is a potent activator of the glycolytic enzyme 6-phosphofructokinase-1 and a strong inhibitor of the gluconeogenic enzyme fructose 1,6-biphosphatase. This indicates that fructose 2,6-biphosphate is a key metabolic signal in the regulation of trehalose synthesis in insects. Trehalose is hydrolysed by trehalase (E.C. 3.2.1.28). The activity of this enzyme is regulated in flight muscle, but the mechanism by which this is achieved is unknown. Trehalase from locust flight muscle is a glycoprotein bound to membranes of the microsomal fraction. The enzyme can be activated by detergents in vitro and by short flight intervals in vivo, which indicates that changes in the membrane environment modulate trehalase activity under physiological conditions.

Amino Acid Sequence↗
PubMed1997

Continuous cultivation of bakers' yeast: change in cell composition at different dilution rates and effect of heat stress on trehalose level.

The cell composition of bakers' yeast in a continuous culture was determined for different dilution rates. Also, the cellular response to heat stress in terms of trehalose, RNA, glycogen and protein was determined at a specified dilution rate of 0.1/h. The amount of storage saccharides, trehalose and glycogen, was found to decrease whereas the amount of RNA and protein increased with increasing dilution rates. As the dilution rate was increased from 0.1 to 0.4/h at 0.05 intervals the steady-state trehalose content decreased from 33 to 8.6 mg/g biomass, and glycogen content from 150 to 93 mg/g biomass. On the other hand, the protein content increased from 420 to 530 mg/g biomass and the RNA content from 93 to 113 mg/g biomass. Heat stress was applied by increasing the medium temperature from 30 to 36, 38 or 40 degrees C at constant dilution rates. The highest amount of trehalose accumulation, 108 mg/g biomass, was achieved when heat stress at 38 degrees C was applied. The protein content, on the other hand, decreased from 350 to 325 mg/g biomass at the end of the experiment.

Biomass↗
PubMed2003

Trehalose, glycogen and ethanol metabolism in the gcr1 mutant of Saccharomyces cerevisiae.

Since Gcr1p is pivotal in controlling the transcription of glycolytic enzymes and trehalose metabolism seems to be one of the control points of glycolysis, we examined trehalose and glycogen synthesis in response to 2% glucose pulse during batch growth in gcr1 (glucose regulation-1) mutant lacking fully functional glycolytic pathway and in the wild-type strain. An increase in both trehalose and glycogen stores was observed 1 and 2 h after the pulse followed by a steady decrease in both the wild-type and the gcr1 mutant. The accumulation was faster while the following degradation was slower in gcr1 cells compared to wild-type ones. Although there was no distinct glucose consumption in the mutant cells it seemed that the glucose repression mechanism is similar in gcr1 mutant and in wild-type strain at least with respect to trehalose and glycogen metabolism.

Culture Media↗
PubMed2002

Trehalose degradation and glucose efflux precede cell ejection during germination of heat-resistant ascospores of Talaromyces macrosporus.

Talaromyces macrosporus forms ascospores that survive pasteurization treatments. Ascospores were dense (1.3 g ml(-1)), relatively dry [0.6 g H(2)O (g dry weight)(-1)] and packed with trehalose (9-17% fresh weight). Trehalose was degraded to glucose monomers between 30 and 100 min after heat activation of the spores. The maximal activity of trehalase was calculated as 400-520 nmol glucose formed min(-1) (mg protein)(-1) as judged by measurements of the trehalose content of spores during germination. During early germination, glucose was released from the cell (10% of the cell weight or more). The intracellular concentration of glucose only peaked briefly. After 160-200 min, the protoplast encompassed by the inner cell wall was ejected through the outer cell wall in a very quick process. Subsequently, respiration of spores increased strongly. The data suggested that trehalose is primarily present for the protection of cell components as glucose is released from the cell. Then, an impenetrable outer cell wall is shed before metabolic activity increases.

Ascomycota↗
PubMed1998

Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli.

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from alpha-D-glucose 1-phosphate and D-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the data-bases. Recombinant TSase with a histidine tag at the NH2-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of alpha-D-glucose 1-phosphate and D-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis.

Amino Acid Sequence↗
PubMed2002

Effects of pre-exercise ingestion of trehalose, galactose and glucose on subsequent metabolism and cycling performance.

The glycaemic and insulinaemic responses to different carbohydrates vary and these have been suggested to affect performance. The purpose of the present study was to determine the effects of pre-exercise ingestion of glucose (GLU), galactose (GAL) and trehalose (TRE) on metabolic responses at rest and during exercise and on subsequent time-trial (TT) performance. Eight well-trained male cyclists completed three exercise trials separated by at least 3 days. At 45 min before the start of exercise subjects consumed 500 ml of a beverage containing 75 g of either glucose, galactose or trehalose. The exercise trials consisted of 20 min of submaximal steady-state exercise (SS) at 65% of maximal power output immediately followed by a [mean (SEM)] 702 (25) kJ TT. Plasma glucose concentration 15 min postprandial was significantly higher in GLU compared to GAL and TRE ( P<0.05). This was accompanied by a more than twofold greater rise in plasma insulin concentration in GLU compared to GAL and TRE (118% and 145%, respectively). During SS exercise four subjects in GLU and one subject in TRE developed a rebound hypoglycaemia (plasma glucose concentration less than 3.5 mmol.l(-1)). No differences were observed in TT performance between the three trials. Pre-exercise ingestion of trehalose and galactose resulted in lower plasma glucose and insulin responses prior to exercise and reduced the prevalence of rebound hypoglycaemia. Despite the attenuated insulin and glucose responses at rest and during exercise following pre-exercise ingestion of galactose and trehalose, there was no difference in TT performance compared with pre-exercise ingestion of glucose.

Adult↗
PubMed2002

A novel thermophilic fusion enzyme for trehalose production.

In recent years a number of hyperthermophilic micro-organisms of Sulfolobales have been found to produce trehalose from starch and dextrins. In our laboratory genes encoding the trehalosyl dextrin forming enzyme (TDFE) and the trehalose forming enzyme (TFE) of S. solfataricus MT4 have been cloned and expressed in E. coli (Rb791). Here we report the construction of a new protein obtained by fusion of TFE and TDFE coding sequences which is able to produce trehalose from dextrins at high temperature by sequential enzymatic steps. We demonstrate that the bifunctional fusion enzyme is able to produce trehalose starting from malto-oligosaccharides at 75 degrees C. Furthermore we partially purified the recombinant fusion protein from bacterial cell free extracts and from insoluble fractions in which the fusion protein was also found as aggregate in inclusion bodies.

Bacterial Proteins↗
PubMed2007

Thermal destabilization of stem bromelain by trehalose.

Trehalose, a naturally occurring osmolyte, is considered as a universal protein stabilizer. We investigated the effect of the disaccharides, trehalose and sucrose, on the thermal stability and conformation of bromelain. To our surprise, bromelain in the presence of 1 M trehalose/sucrose was destabilized under thermal stress. The average Tm values as determined by UV spectroscopy and CD spectropolarimetry decreased by 5 degrees and 7 degrees C for bromelain in 1 M sucrose or trehalose solutions, respectively. The enzyme was also found to inactivate faster at 60 degrees C in the presence of these osmolytes. The tertiary and secondary structure of bromelain undergoes small changes in the presence of sucrose/trehalose. Studies on the binding of these osmolytes with the native and the heat denatured enzyme revealed that sucrose/trehalose lead to preferential hydration of the denatured bromelain as compared to the native one, hence stabilizing more the denatured conformation. This is perhaps the first report on the destabilization of a protein by trehalose.

Ananas↗
PubMed1985

Preservation of freeze-dried liposomes by trehalose.

One of the practical difficulties with the frequently proposed use of liposomes for delivery of water-soluble substances to cells in whole organisms is that liposomes are relatively unstable during storage. We have studied the ability of trehalose, a carbohydrate commonly found at high concentrations in organisms capable of surviving dehydration, to stabilize dry liposomes. With trehalose both inside and outside the bilayer, almost 100% of trapped solute was retained in rehydrated vesicles previously freeze-dried with 1.8 g trehalose/g dry phospholipid. Trehalose is very effective at inhibiting fusion between liposomes during drying, as assessed by freeze-fracture and resonance energy transfer between fluorescent probes incorporated into the bilayer. However, inhibition of fusion alone does not account for the preservation of the dry liposomes, since the concentration of trehalose required to prevent leakage is more than 10-fold that required to prevent fusion. We provide evidence that stabilization of the dry liposomes requires depression of transition temperature and consequent maintenance of the constituent lipids in the dry liposomes in a liquid crystalline phase.

Chromatography, Gel↗
PubMed1990

Synthesis of a trehalose homolog, 6-deoxy-alpha-D-gluco-heptopyranosyl 6-deoxy-alpha-D-gluco-heptopyranoside, and the corresponding bis(heptosiduronic acid).

Crystalline 6-deoxy-alpha-D-gluco-heptopyranosyl 6-deoxy-alpha-D-gluco- heptopyranoside (8) and (6-deoxy-alpha-D-gluco-heptopyranosyluronic acid) 6-deoxy-alpha-D-gluco-heptopyranosiduronic acid (7) were synthesized from alpha, alpha-trehalose (1). Reaction of 2,3,4,2',3',4'-hexa-O-acetyl- 6,6'-di-O-tosyl-alpha, alpha-trehalose with sodium dicarbonylcyclopentadienyliron, followed by oxidative hydrolysis or methanolysis, gave the 2,3,4,2',3',4'-hexa-acetate of 7 or its dimethyl ester, respectively. O-Deacetylation (Zemplén) then gave 7 and its dimethyl ester. Reduction of the hexa-O-acetyldicarboxylic acid with borane-oxolane complex yielded 8. Alternatively, cyanide displacement of hexa-O-acetyl-alpha, alpha-trehalose 6,6'-ditriflate gave the dinitrile hexa-acetate of 7, which was O-deacetylated and then hydrolyzed with alkaline hydrogen peroxide to yield 7. 2,3,4,2',3',4'-Hexa-O-benzyl-alpha, alpha-trehalose 6,6'-ditriflate was similarly converted into the dinitrile, which was hydrolyzed to the corresponding diamide. Treatment of the 2,3,4,2',3',4'-hexa- O-acetyl-alpha, alpha-trehalosuronic acid 16 with thionyl chloride followed by diazomethane gave a crystalline bisdiazoketone which, however, failed to produce the expected bis(heptosiduronic acid) on attempted Wolff rearrangement.

Acetylation↗
PubMed1990

Regioselective syntheses of trehalose-containing trisaccharides using various glycohydrolases.

Two methods were investigated for the enzymic preparation of trehalose-containing trisaccharides. In the first, a solution of saccharides is circulated through an immobilized-glycosidase column and an activated-carbon column connected in series. In the second, two enzymes having different substrate specificities are sequentially used for condensation and subsequent specific hydrolysis. Thus, 3-O-beta-D-, 4-O-beta-D-, and 6-O-beta-D-galactopyranosyl-alpha,alpha-trehalose; 4-O-alpha-D- and 6-O-alpha-D-glucopyranosyl-alpha,alpha-trehalose; and 4-O-beta-D- and 6-O-beta-D-glucopyranosyl-alpha,alpha-trehalose were synthesized stereo- and regio-selectively.

Antigens, Bacterial↗
PubMed1988

Cryopreservation of murine embryos with trehalose and glycerol.

Several concentrations of trehalose (0.0, 0.04, 0.1, 0.25 M) in combination with three concentrations of glycerol (1.0, 1.5, 2.0 M) were evaluated for the cryopreservation of murine embryos. Embryos were transferred through increasing concentrations of glycerol in Dulbecco's phosphate-buffered saline with 10% fetal calf serum (PBS + FCS) to reach the final glycerol concentrations. They were then randomly assigned to one of the concentrations of trehalose. A total of 506 morulae were packaged individually in 0.25-ml plastic straws and cooled from ambient temperature at 1.0 degrees C/min in a programmable methanol freezer. Embryos were seeded at -7 degrees C and then cooled to -25 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. After thawing and a one-step dilution of glycerol, embryos were cultured for 48 hr and viability was determined by blastocoel formation. Highest viability (70.0%) after 48 hr in culture was obtained for embryos frozen in 1.5 M glycerol plus 0.10 M trehalose as compared to 31% viability for embryos frozen with glycerol alone. These observations suggest that trehalose can be used in combination with glycerol as a cryoprotectant and that a high rate of viability can be achieved after a one-step dilution of the cryoprotectants.

Animals↗
PubMed1991

Effects of heat shock on the level of trehalose and glycogen, and on the induction of thermotolerance in Neurospora crassa.

Neurospora crassa conidiospore germlings exposed to a heat shock (30-45 C) rapidly accumulated trehalose and degraded glycogen, even in the presence of cycloheximide. This phenomenon was also rapidly reversible upon return of the cells at 30 degrees C. Trehalose accumulation at 45 degrees C demanded an exogenous source of carbon and either glucose or glycerol fulfilled such requirement. Experiments with the cyclic AMP-deficient cr-1 mutant suggested that the effects of temperature shifts on trehalose level were independent of cAMP metabolism. Cells exposed at 45 degrees C under conditions permissive for trehalose accumulation (i.e. in the presence of an assimilable carbon source) also acquired thermotolerance.

Cyclic AMP↗