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PubMed1975

Some properties of trehalase from Phycomyces blakesleeanus.

Trehalase (alpha, alpha-trehalase glucohydrolase EC 3.2.1.28) from Phycomyces spores occurs in two different forms which are convertible in vivo: a form with low activity found in dormant spores and an active form after breaking the dormancy. Between the two forms no difference in molecular weight and electrophoretic mobility can be detected. The molecular weight is estimated by gel filtration at about 210 000. The relation between substrate concentration and trehalase activity follows the Michaelis-Menten equation (K-m plus or minus 55 mM) in activated spores whereas in dormant spores trehalase shows a different substrate binding, indicating a negative cooperative effect. They differ further in thermostability and in sensitivity to inhibition by ATP. Other nucleosidephosphates have no inhibiting effect. Heating the spores at different temperatures between 38 and 44 degrees C results in a partial breaking of dormancy of the spore population and a corresponding partial activation of trehalase. This suggests a close connection between breaking dormancy and trehalase activation.

Chromatography, Gel↗
PubMed1989

20-Hydroxyecdysone acts in the male pupa to commit accessory glands toward trehalase production in the adult mealworm beetle (Tenebrio molitor).

During postecdysial adult maturation, the bean-shaped accessory reproductive glands (BAGs) of adult male mealworm beetles produce increasing amounts of trehalase. In order to determine when the BAGs become competent to produce trehalase, we transplanted pupal BAGs into 0-day female adults. After 8 days, trehalase activity had increased in BAGs from 4- and 5-day pupae (at the time of the pupal ecdysteroid peak) but not in those from 1- and 2-day pupae (before the ecdysteroid peak). BAGs from 0- and 2-day pupae were exposed to 20-hydroxyecdysone in vitro before implantation into 0-day female adults. Increase in trehalase activity was dose dependent. Both dose (ED50, 5 x 10(-6) M) and exposure time (greater than 6 hr) of hormone required are greater for commitment than for acceleration of pupal cell cycling (T. Yaginuma, H. Kai, and G.M. Happ, 1988, Dev. Biol. 126, 173-181). Since trehalase activity increased markedly in isolated adult male abdomens, factors from the cephalic and thoracic centers are not required to sustain trehalase production in the adult BAGs.

Abdomen↗
PubMed1994

Molecular characterization of ovary trehalase of the silkworm, Bombyx mori and its transcriptional activation by diapause hormone.

We have isolated a cDNA encoding ovary trehalase of the silkworm, Bombyx mori. Sequence analyses revealed that the isolated cDNA contains 3143 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis showed a 3.0 kb transcript in developing ovaries carrying membrane-bound trehalase. A single copy of trehalase gene was present in the haploid genome of the silkworm. The effect of diapause hormone on the accumulation of trehalase mRNA was examined on developing ovaries in in vivo and in vitro conditions. The synthetic diapause hormone brought about a 6-fold increase in trehalase mRNA content in ovaries 4 h after injection. The similar increase was found in ovaries which were incubated in vitro with diapause hormone. Coincubation of ovaries with diapause hormone and actinomycin D could not increase the mRNA level in ovaries, and maintained a basal level which was found in ovaries incubated without diapause hormone. These results indicate that diapause hormone stimulates transcription of the trehalase gene in developing ovaries of the silkworm.

Amino Acid Sequence↗
PubMed1986

Purification and characterization of amphiphilic trehalase from rabbit small intestine.

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.

Animals↗
PubMed1997

Osmo-stress-induced changes in neutral trehalase activity of the fission yeast Schizosaccharomyces pombe.

Exposure of repressed growing cultures of Schizosaccharomyces pombe to various extracellular concentrations of NaCl, sorbitol or glycerol resulted in a reversible increase in neutral trehalase activity which was maintained while the cells were in the presence of high environmental osmolarity. Treatment of osmo-stress-induced trehalase by phosphatase lead to a decreased activity indicating that the active enzyme is phosphorylated. The stress response following the osmotic shock required protein synthesis and was independent of the cAMP-dependent protein kinase pathway. Cells disrupted for wis] or phh1 (identical to sty1 and spc1), which encode members of the mitogen-activated protein kinase (MAPK) cascade, showed that the osmo-stress-induced increase in trehalase markedly diminished. In contrast, the heat shock-induced increase in trehalase remained unchanged in these cells. Taken together, the data suggest that the elevation of trehalase activity in Schiz. pombe under conditions of high osmolarity is due to de novo synthesis of the enzyme and that this process is modulated through a MAPK signal transduction pathway as part of the physiological response to the osmotic stress. The wisl-phhl MAPK cascade, however, does not appear to form part of the mechanism underlaying the increase in trehalase after heat stress.

Hot Temperature↗
PubMed2001

Cloning, characterization and mapping of the mouse trehalase (Treh) gene.

Trehalase is the least studied of the membrane-bound alpha- glucosidase enzymes. Here we report the isolation and characterization of the mouse trehalase (Treh) gene. Initially, PCR using primers based on published rat cDNA sequence was used to clone a partial mouse cDNA. This allowed design of mouse primers which identified a single positive clone in a bacterial artificial chromosome (BAC) library of mouse genomic DNA. Analysis of BAC subclones showed that the Treh structural gene spans approximately 13 kb and comprises 15 exons. Data from genomic Southern blotting were consistent with mouse Treh being a single copy gene. The transcription initiation site was determined by both S1 nuclease mapping and 5' rapid amplification of cDNA ends (5' RACE) to be located 25 nt upstream of the ATG in exon 1. The mouse Treh exons were found to have an open reading frame of 1728 nt and the encoded protein of 576 amino acids showed 81, 82 and 93% amino acid sequence identity with rabbit, human and rat trehalase, respectively. The trehalase signature sequence found at amino acids 162 to 175 had 100% identity with the corresponding region of rabbit, human and rat and 79% identity with that for yeast trehalase. When a mouse Treh cDNA was used for Northern blot analysis of RNA from 12 mouse tissues, Treh mRNA expression was detected only in kidney and small intestine. The size of the mRNA in both of these tissues was estimated to be approximately 2.1 kb, furthermore both tissues appear to have the same transcription initiation site as determined by nuclease protection. Using the T31 radiation hybrid panel, mouse Treh was shown to be located on Chromosome 9 in a broad region that is orthologous with human Chromosome 11q23. The human trehalase gene (TREH) was identified in the latter location via database searching, which also revealed the overall structure of the human gene as being similar to that of the mouse.

Amino Acid Sequence↗
PubMed2000

Changes in external trehalase activity during human serum-induced dimorphic transition in Candida albicans.

Yeast-like cells (blastoconidia) of Candida albicans growing exponentially on a glucose-containing medium (YPD) exhibited low external trehalase activity and stored a negligible amount of intracellular trehalose. The addition of human serum at 37 degrees C to exponential cultures promoted a high degree of germ-tube formation with no significant changes in trehalase activity or trehalose content. In contrast, stationary cells accumulated a large amount of trehalose, while external trehalase remained at a low and practically constant level. However, resting cultures were unable to enter the dimorphic program, except when they were supplemented with fresh YPD and serum together. Only under these conditions was trehalase activated and trehalose hydrolyzed. Specific inhibition of external trehalase by validoxylamine A caused a certain delay in, and a lower level of, germ-tube formation, but did not totally block the dimorphic conversion. These results suggest that external trehalase is not involved in the serum-induced morphological transition in C. albicans.

Blood Proteins↗
PubMed1982

Trehalase: stereocomplementary hydrolytic and glucosyl transfer reactions with alpha- and beta-D-glucosyl fluoride.

A new understanding has been obtained of the catalytic capabilities of trehalase, an enzyme heretofore held to be strictly specific for hydrolyzing alpha, alpha-trehalose and devoid of transglycosylative ability. Highly purified rabbit renal cortical trehalase and a partly purified Candida tropicalis yeast trehalase were found to utilize both alpha- and beta-D-glucosyl fluoride as substrates. In each case, the reactions were competitively inhibited by alpha, alpha-trehalose. Both enzymes catalyzed rapid hydrolysis of alpha-D-glucosyl fluoride to form beta-D-glucose (also, of alpha, alpha-trehalose to form equimolar alpha- and beta-D-glucose). In addition, digests of beta-D-glucosyl fluoride plus alpha-D-[14C]-glucopyranose with either trehalase (but not controls of enzyme with alpha-D-[14C]glucopyranose alone) yielded small amounts of radioactive trehalose (alpha-D-glucopyranosyl alpha-D-[14C]glucopyranoside) which does not accumulate since it is rapidly hydrolyzed. Trehalase thus catalyzes two stereocomplementary types of glycosylation reactions: (I) alpha-D-glucosyl fluoride (or alpha, alpha-trehalose) + H2O leads to beta-D-glucose + HF (or alpha-D-glucose); (II) beta-D-glucosyl fluoride + alpha-D-glucopyranose leads to alpha, alpha-trehalose + HF. Such behavior shows that the catalytic groups of trehalase, as recently found for other glycosylases, are functionally flexible. The results illustrate the inadequacy of conventional views of carbohydrase specificity and the rigor, as a basic guiding principle, of the concept that glycoside hydrolases and glycosyltransferases form a class of glycosylases effecting glycosyl/proton interchange.

Animals↗
PubMed1992

Trehalase from male accessory gland of an insect, Tenebrio molitor. cDNA sequencing and developmental profile of the gene expression.

A cDNA of alpha alpha-trehalase (EC 3.2.1.28) from a cDNA library of male bean-shaped accessory gland of the mealworm beetle, Tenebrio molitor, has been isolated by the homology screening approach. Sequence analysis of the cDNA (1830 bp) revealed that the cDNA encoded a protein of 555 amino acids with a calculated M(r) of 64457. The deduced amino acid sequence had significant similarities to rabbit small intestine and Escherichia coli trehalases. Northern blotting and semi-quantitative PCR analyses revealed that a trehalase transcript with about 2.0 kb was abundant in bean-shaped accessory glands. In the glands, the amount of trehalase transcript increased from 1 to 2 days after adult ecdysis. These tissue- and stage-specific gene expressions of trehalase corresponded to the tissue- and stage-specificity of trehalase activity.

Amino Acid Sequence↗
PubMed2002

Molecular interaction of neutral trehalase with other enzymes of trehalose metabolism in the fission yeast Schizosaccharomyces pombe.

Trehalose metabolism is an essential component of the stress response in yeast cells. In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1+ (coding for trehalose-6-P synthase, Tps1p), ntp1+ (encoding neutral trehalase, Ntp1p) and tpp1+ (that codes for trehalose-6-P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes. Disruption of the gene tps1+ blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress. We propose that this association may reflect the Tps1p-dependent requirement for thermal activation of trehalase. Data reported here indicate that following a heat shock the enzyme activity of trehalase is associated with Ntp1p dimers or trimers but not with either Ntp1p monomers or with complexes involving Tps1p. These results raise the possibility that heat shock and osmotic stress activate trehalase differentially by acting in the first case through an specific mechanism involving Tps1p-Ntp1p complexes. This study provides the first evidence for the participation of the catabolic enzyme trehalase in the structural framework of a regulatory macromolecular complex containing trehalose-6-P synthase in the fission yeast.

Chromatography, Gel↗
PubMed1989

Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori.

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.

Alkaline Phosphatase↗
PubMed2002

Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae.

Various microorganisms produce the disaccharide trehalose during their symbiotic and pathogenic interactions with plants. Trehalose has strong effects on plant metabolism and growth; therefore, we became interested to study its possible role in the interaction of Arabidopsis thaliana with Plasmodiophora brassicae, the causal agent of clubroot disease. We found that trehalose accumulated strongly in the infected organs (i.e., the roots and hypocotyls) and, to a lesser extent, in the leaves and stems of infected plants. This accumulation pattern of trehalose correlated with the expression of a putative trehalose-6-phosphate synthase (EC 2.4.1.15) gene from P. brassicae, PbTPS1. Clubroot formation also resulted in an induction of the Arabidopsis trehalase gene, ATTRE1, and in a concomitant increase in trehalase (EC 3.2.1.28) activity in the roots and hypocotyls, but not in the leaves and stems of infected plants. Thus, induction of ATTRE1 expression was probably responsible for the increased trehalase activity. Trehalase activity increased before trehalose accumulated; therefore, it is unlikely that trehalase was induced by its substrate. The induction of trehalase may be part of the plant's defense response and may prevent excess accumulation of trehalose in the plant cells, where it could interfere with the regulation of carbon metabolism.

Amino Acid Sequence↗
PubMed1985

Alterations in trehalase solubility during development in the cellular slime mould Dictyostelium discoideum.

Previous studies have indicated that during development in the slime mould Dictyostelium discoideum, compartmentation of the isoenzymes of trehalase (alpha, alpha'-trehalose 1-D-glucohydrolase, (EC 3.2.1.28) occurs between the extracellular and intracellular environments. The compartmentation of trehalase between soluble and particulate cell fractions was examined in this work. The trehalase present in crude homogenates prepared during the first 12 h of development was completely soluble. Starting at about the pseudoplasmodial stage (i.e. the 14th hour of development), trehalase activity became associated with insoluble cellular material and this increased to a maximal value in homogenates from mature sorocarps, where 50% of the activity was insoluble. Spore cells accounted for only 2 to 3% of the trehalase associated with mature sorocarps, with the remaining 97% being localized in stalk cell material. Although trehalase recovered from spores was completely soluble, more than half of that from the stalk was recovered in the buffer-insoluble pellet fraction.

Cell Compartmentation↗
PubMed1988

Regulation of trehalase activity during the cell cycle of Saccharomyces cerevisiae.

Synchronous cultures of Saccharomyces cerevisiae prepared by selection of small unbudded cells from an elutriating rotor were used to measure trehalase activity during the cell cycle. After the small cells had been removed from the rotor, the remainder was used to prepare asynchronous control cultures. Both synchronous and control cultures were studied for two cell cycles. In asynchronous cultures the trehalase activity of crude cell lysates rose continuously. In synchronized populations trehalase activity increased from the beginning of budding onwards. However, around the period of cell division the enzyme activity dropped rapidly but transiently by more than 5-fold. The same changes were found during the second budding cycle. Measurements of invertase and glucose-6-phosphate dehydrogenase activities in the same synchronous and asynchronous cultures revealed a continuous increase for both enzymes. Incubation of cell lysates with cAMP-dependent protein kinase before assaying for trehalase resulted in a 2-fold enhancement of enzyme activity in asynchronous control cultures. In synchronized cells this treatment also led to a significant stimulation of trehalase activity, and largely abolished the cell-cycle-dependent oscillatory pattern of enzyme activity. These results suggest that the activity of trehalase during the cell cycle is regulated, presumably at the post-translational level, by a phosphorylation-dephosphorylation mechanism.

Cell Cycle↗
PubMed1992

Nutrient-induced activation of trehalase in nutrient-starved cells of the yeast Saccharomyces cerevisiae: cAMP is not involved as second messenger.

Starvation of Saccharomyces cerevisiae cells for specific nutrients such as nitrogen, phosphate or sulphate causes arrest in the G1 phase of the cell cycle at a specific point called 'start'. Re-addition of different nitrogen sources, phosphate or sulphate to such starved cells causes activation of trehalase within a few minutes. Nitrogen-source- and sulphate-induced activation of trehalase were not associated with any change in the cAMP level, but in the case of phosphate there was a small transient increase. When nitrogen-source-activated trehalase was isolated by immuno-affinity chromatography from crude extracts, the purified enzyme showed the same activity profile as in the original crude extracts, indicating that post-translational modification is responsible for the activation. In the yeast mutants cdc25-5 and cdc35-10, which are temperature sensitive for cAMP synthesis, incubation at the restrictive temperature lowered but did not prevent nitrogen-, phosphate- or sulphate-induced activation of trehalase. Since under these conditions the cAMP level in the cells is very low, it is unlikely that cAMP acts as a second messenger in this nutrient-induced effect. Nitrogen-source-induced activation of trehalase requires the presence of glucose at a concentration similar to that able to stimulate the RAS-adenylate cyclase pathway. This indicates that the same glucose-sensing system might be involved in both phenomena. Nitrogen-starved cells fractionated according to cell size all showed nitrogen-source-induced activation of trehalase to the same extent, indicating that the nitrogen-induced signalling pathway involved is not dependent on the well-known cell size requirement for progression over the start point of the cell cycle.

Ammonium Chloride↗
PubMed1983

Reversibility characteristics of glucose-induced trehalase activation associated with the breaking of dormancy in yeast ascospores.

The breaking of dormancy in yeast ascospores by addition of glucose is associated with a sudden tenfold increase in the activity of trehalase. The rapid activation of trehalase is followed by a slower inactivation process which is greatly retarded in the presence of nitrogen sources and cycloheximide. When glucose is washed away from the spores after some time and the spores resuspended in glucose-free medium, the trehalase activity decreases sharply. Subsequent addition of new glucose partially reactivates the enzyme. The extent of reactivation decreases further with each subsequent activation/inactivation step. Changing the duration of the inactivation periods has no effect on this diminution of the reversibility. However, prolonging the duration of the activation step speeds up the loss of reversibility. On the other hand, addition of a nitrogen source or cycloheximide completely prevents the loss of reversibility. The results of the reversibility studies are in agreement with the phosphorylation mechanism which has been proposed for the underlying molecular process of trehalase activation. Apparently, they are also in agreement with proteolytic breakdown being responsible for the inactivation of trehalase after its initial activation. However, the effect of cycloheximide and nitrogen sources, at least in ascospores, does not appear to be due to inhibition or repression of protease synthesis, respectively, since the addition in the presence of glucose of a nitrogen source after trehalase inactivation immediately reactivates the enzyme completely.

Ascomycota↗
PubMed1997

Trehalases and trehalose hydrolysis in fungi.

The simultaneous presence of two different trehalose-hydrolysing activities has been recognised in several fungal species. While these enzymes, known as acid and neutral trehalases, share a strict specificity for trehalose, they are nevertheless rather different in subcellular localisation and in several biochemical and regulatory properties. The function of these apparently redundant activities in the same cell was not completely understood until recently. Biochemical and genetic studies now suggest that these enzymes may have specialised and exclusive roles in fungal cells. It is thought that neutral trehalases mobilise cytosolic trehalose, under the control of developmental programs, chemical and nutrient signals, or stress responses. On the other hand, acid trehalases appear not to mobilise cytosolic trehalose, but to act as 'carbon scavenger' hydrolases enabling cells to utilise exogenous trehalose as a carbon source, under the control of carbon catabolic regulatory circuits. Although much needs to be learned about the molecular identity of trehalases, it seems that in fungi at least one class of acid trehalases evolved independently from the other trehalases.

Fermentation↗
PubMed2000

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe.

Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.

Culture Media↗