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PubMed1973

Localization of trehalase in the ascospores of Neurospora: relation to ascospore dormancy and germination.

An association of trehalase with the innermost wall (endosporium) of ascospores of Neurospora is suggested, because this enzyme could be lyophilized in the presence of various wall components and heated in this dried state at 65 C without loss of activity. Ground ascospore walls, purified mycelial walls, a wall fraction consisting of protein, glucan and polygalactosamine, or bovine serum albumin stabilize trehalase under these conditions. No other substances tested protected as well as the above materials. Immunofluorescent labeling of trehalase shows that it is localized in the endosporium. Therefore, it is most probable that in dormant ascospores of Neurospora, trehalase, and its substrate, trehalose, are physically separated. Trehalose is located in the cytoplasm, whereas trehalase resides within the protein and carbohydrate matrix of the innermost major cell wall layer of the ascospore. The association with the cell wall protects the enzyme against the heating which is necessary to activate germination. Activation, whether by heat or chemical treatment (furfural), probably involves an increase in the permeability of the ascospore plasma membrane allowing trehalose to diffuse to the vicinity of its hydrolase, thereby providing the energy and intermediates for germination.

Antigens, Fungal↗
PubMed2003

Routine use of a one minute trehalase and maltase test for the identification of Candida glabrata in four laboratories.

AIMS: To evaluate the rapid identification of Candida glabrata using a one minute trehalase and maltase test in four clinical laboratories. METHOD: The test was evaluated with 944 freshly isolated yeasts comprising 572 C glabrata and 372 non-C glabrata strains. These strains were isolated on one of three differential media-Candida ID, CHROMagar Candida, or Albicans ID2 medium-and all strains were fully identified using standard methods. RESULTS: The trehalase and maltase test allowed the overall identification of 550 of 572 C glabrata strains (sensitivity, 96.2%) and only 11 of 372 isolates of other yeast species yielded a false positive result (specificity, 96.8 %). Sensitivity and specificity were consistent from one laboratory to another. Using Candida ID medium, the rapid trehalase and maltase test showed a sensitivity of 95% and specificity of 96.2%. Using CHROMagar Candida, sensitivity and specificity were 95.6% and 98.1%, respectively. Using Albicans ID2 medium (tested by two laboratories), the sensitivity was 100% and 98.5% and specificity was 98.1% and 98.2%. In 60% of cases, the test could be performed directly from the primary isolation medium, thus reducing the time for identification. CONCLUSION: The rapid trehalase and maltase test was highly reliable for the presumptive identification of C glabrata on primary isolation using three different chromogenic media. Direct recognition of C albicans by means of their characteristic colour on chromogenic media coupled with one minute trehalase maltase testing performed only on suspect colonies of C glabrata allowed for rapid presumptive identification of the two yeast species most commonly encountered in clinical samples.

Candida glabrata↗
PubMed1981

Trehalase activity in genetically diabetic mice (serum, kidney, and liver).

Trehalase activity was determined in serum, liver, and kidney in alloxan treated Swiss mice and in homozygous (Ob/Ob, Db/Db) and heterozygous (Ob/+, Db/m+) diabetic mice. Both alloxan and genetic diabetic mice exhibited a large increase in serum and liver trehalase activity with no change in kidney trehalase activity. The heterozygotes (Ob/+, Db/m+) showed only a slight increase of enzyme activity. Further quantitative differences were noticed between the genetic and alloxan diabetic animals. The liver enzyme activity increased from 10- to more than 20-fold in the liver of the homozygous Ob/Ob and Db/Db strains and only 3-fold (not significant compared to controls) in the alloxan treated animals. The above results suggest a regulatory relationship between the genes coding for trehalase and the enzymes of glucose metabolism activity involved in the development of the metabolic anomalies of diabetes. The structural gene for trehalase may well have survived elimination of selective pressure during phylogenesis and remained part of a co-regulated group of glucose metabolising enzymes. This could explain its sensitivity to mutations affecting glucose metabolism and its sensitivity to insulin directed regulatory mechanisms.

Animals↗
PubMed1988

Transport of yeast vacuolar trehalase to the vacuole.

We have tested yeast secretory mutants, which define different stages of the secretory pathway, for their levels of vacuolar trehalase activity. Mutations that cause accumulation of secretory proteins in the endoplasmic reticulum or in the Golgi body lead to diminished vacuolar trehalase activity. Mutations that cause accumulation of secretory vesicles have no effect on vacuolar trehalase activity. None of the mutations affects cytoplasmic trehalase activity. These results provide further evidence for the existence of a compartmentalized trehalase in yeast, and demonstrate that the enzyme enters the secretory pathway.

Biological Transport↗
PubMed1979

The localization of honey bee thorax trehalase.

Differential and sucrose gradient centrifugation of honey bee thoraces, disrupted by gentle methods and using mannitol-triethanolamine-EDTA buffer at pH 6.5, showed that in the honey bee thorax 92-94.8% of the trehalase was mitochondrial. Since only 92-95% of the cytochrome c oxidase, a known mitochondrial enzyme, was found in the mitochondrial fraction by these methods, it was concluded that honey bee trehalase is totally mitochondrial. Significant amounts of 'microsomal' or 'soluble' trehalase were formed only by harsh methods of thorax disruption and similar 'microsomal' or 'soluble' trehalases were also formed by harsh treatment of purified whole mitochondria. They thus seem to be artifacts of the isolation procedure. Studies (using marker enzymes) with purified intact mitochondria which were dispersed by various chemical, enzymatic, and physical methods showed that the trehalase in the mitochondria was membrane bound and that it was bound to either the outside of the inner membrane or to one of the sides of the outer membrane.

Animals↗
PubMed1988

Increased trehalase expression after glucose limitation of LLC-PK1 clones.

Expression of the apical membrane disaccharidase trehalase correlates with the development of a proximal tubule-specific differentiated phenotype in the LLC-PK1 cell line derived from renal epithelia. Substitution of galactose for glucose in the culture medium of confluent cultures resulted in a significant trehalase induction; a smaller induction was observed after replacement by fructose, mannose, or the nonmetabolizable sugar alpha-methyl-D-glucose. Induction was specific for trehalase among other microvillar hydrolases, reversible after replacement of glucose, and was increased as a function of decreasing glucose concentration. Stable clonal cell lines that were inducible by glucose limitation and noninducible clones were isolated nonselectively. Both types exhibited trehalase induction after exposure to the differentiation inducer N,N'-hexamethylene-bis-acetamide (HMBA). These results suggest that trehalase induction after glucose deprivation occurs by a different mechanism than that mediated by HMBA.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Urinary trehalase activity is a useful marker of renal proximal tubular damage in newborn infants.

To clarify the reliability of urinary trehalase activity as a marker of cellular proliferation and/or damage of renal proximal tubules, the activity was examined in healthy newborn infants or infants treated with tobramycin, a drug known as causing tubular cell damage. Eighty-one newborn infants (56 mature infants and 25 premature infants) were enrolled in the study. Urinary trehalase was examined using a spot urine sample during the first 7 days of age and on the 10th day of age. A good positive correlation was observed between urinary trehalase activity/creatinine ratio (T/Cr) on the 10th day of age and conceptional age or body weight (n = 46, r = 0.58, p < 0.001). Urinary trehalase of 29 healthy mature infants was higher during the first few days of age, after which it decreased to an almost steady level. Urinary trehalase of 6 premature infants during the first few days of age was significantly lower than that of mature infants, after which it increased and became equal to that of the mature infants on the 7th day of age. Treatment with ampicillin (100 mg/kg) and tobramycin (5 mg/kg) of 6 mature infants with pneumonia for 6 days resulted in a significant elevation of the urinary T/Cr. The extent of this elevation was greater than that of the urinary N-acetyl-beta-D-glucosaminidase (NAG) activity/creatinine ratio (NAG/Cr). A significant correlation was observed between the urinary T/Cr and the urinary NAG/Cr (r = 0.67, p < 0.01) or gamma-glutamyl transpeptidase/creatinine ratio (r = 0.48, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1999

Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae.

A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of neutral trehalase from the yeast ABYS1 mutant.

Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.

Alkaline Phosphatase↗
PubMed1996

Inhibition by polyols of the heat-shock-induced activation of trehalase in the yeast Zygosaccharomyces rouxii.

Trehalase activity was markedly enhanced in Zygosaccharomyces rouxii upon exposure of the cells to a heat shock. The increase in trehalase was independent of rapid changes in the intracellular concentration of cAMP and was not blocked by inhibitors of protein synthesis. Trehalase activated in vivo by heat shock was deactivated in vitro by phosphatase, suggesting that heat stress triggers a cAMP-independent signalling pathway that includes the activation of trehalase by phosphorylation of the enzyme protein. The addition to these cells before heating of either glycerol or other polyols produced a significant decrease in the heat-shock induced activation of trehalase. However, the trehalose content in cells heat-shocked in the presence of polyols did not increase significantly, indicating that these compounds may also influence the synthesis of the disaccharide.

Alkaline Phosphatase↗
PubMed2001

Purification and characterization of an acid trehalase from Acidobacterium capsulatum.

We purified an acid trehalase (EC 3.2.1.28, alpha,alpha'-trehalose glucohydrolase) from an acidophilic bacterium, Acidobacterium capsulatum. The enzyme was homogeneous based on polyacrylamide gel electrophoresis, and was composed of a single polypeptide chain with a molecular mass of 57 kDa. Maximum trehalase activity was observed at pH 2.5. The acid trehalase exhibited an apparent K(m) of 1.0 mM for trehalose at 30 degrees C and pH 3.0. The trehalase was located in the periplasmic space. The activity of the enzyme was activated by 1.0 mM MnCl2 or CoCl2, and inhibited by 1.0 mM PbCl2, HgCl2, NiCl2, p-chloromercuribenzoate, N-ethylmaleimide, monoiodoacetate, or EDTA. The enzyme showed high specificity for trehalose. It was found that an equimolar mixture of alpha-D-glucose and beta-D-glucose was formed on hydrolysis of trehalose by the trehalase.

Journal Article↗
PubMed1991

Characterization of different forms of yeast acid trehalase in the secretory pathway.

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.

Autoradiography↗
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↗
PubMed1988

Trehalase from the bean-shaped accessory glands and the spermatophore of the male mealworm beetle, Tenebrio molitor.

In Tenebrio molitor, male adults transfer sperm to the female via a spermatophore or sperm sac. The spermatophore is formed from secretions of the bean-shaped accessory glands (BAGs) and the tubular accessory glands (TAGs) of the male beetle. Trehalase is found in the adult BAGs. During the pupal stage, the activity in the BAGs was very low. After adult ecdysis, the total activity increased 100-fold from 0 days to 6 days and reached maximum levels at 9 days. The specific activity increased 20-fold from the time of ecdysis to 6 days thereafter. In the 10 day adult, trehalase levels in testes, seminal vesicles, vas deferens, TAGs, or ejaculatory ducts, were lower by two orders of magnitude than in the BAGs. However, the specific activity in the spermatophore was similar to that in the BAGs. Trehalases in the BAGs and the spermatophores showed very similar properties (soluble, optimum pH of 5.75 and Km value of 5.4 mM for trehalose). Thus trehalase appears to be secreted from the BAGs and becomes incorporated into the spermatophores.

Animals↗
PubMed1987

Elevation of urinary trehalase activity in patients of itai-itai disease.

The elevation of urinary trehalase activity in patients of itai-itai disease was examined. Urinary trehalase was correlated with tubular reabsorption of phosphorus (%TRP): the lower the trehalase activity, the worse was %TRP. Furthermore, this activity was inversely correlated with urinary glucose and urinary total protein. In itai-itai disease, the excretion of beta 2-microglobulin seems to be maximal, and urinary trehalase activity was low in the latter stages of the disease. It is inferred that itai-itai disease produces extremely severe tubular damage as well as glomerular dysfunction.

Aged↗
PubMed1989

Analysis and DNA sequence of the osmoregulated treA gene encoding the periplasmic trehalase of Escherichia coli K12.

The treA gene of Escherichia coli K12 codes for a periplasmic trehalase that is induced by growth at high osmolarity. The position of treA within a cloned chromosomal DNA fragment was identified by subcloning of restriction fragments and analysis of the gene product in minicells. The nucleotide sequence of the treA coding region as well as its upstream control region was determined. The treA gene consists of 1695 bp encoding 565 amino acids. The amino-terminus of the mature trehalase was found to begin with the amino acid Glu at position 31 of the open reading frame. The first 30 amino acids resemble a typical signal sequence, consistent with trehalase being a secreted periplasmic enzyme. Two previously isolated phoA fusions to the osmA gene were transferred by homologous recombination on to a treA-containing plasmid and found to be within treA. Analysis of the hybrid genes and their gene products aided the localization of treA and the determination of its direction of transcription within the cloned chromosomal segment. The treA-phoA fusions encoded hybrid proteins which could be found in the periplasm. We found that at high osmolarity the normal pathway for the uptake and utilization of trehalose is blocked. Therefore, the function of the periplasmic trehalase is to provide the cell with the ability to utilize trehalose at high osmolarity by splitting it into glucose molecules that can subsequently be taken up by the phosphotransferase-mediated uptake system.

Amino Acid Sequence↗
PubMed1992

A transfer membrane method for in situ detection and quantification of trehalase.

A method for the detection and quantification of trehalase activity (EC 3.2.1.28) by immobilization to a membrane support has been developed. Protein samples partly enriched for porcine and Galleria mellonella wax moth larvae trehalase activities were fractionated by polyacrylamide gel electrophoresis, followed by electrophoretic transfer to PVDF membranes, and incubated in a solution containing trehalose (20 mg/ml), glucose oxidase (40 U/ml), phenazine methosulfate (0.06 mg/ml), and nitro blue tetrazolium (0.24 mg/ml) in 20 mM sodium phosphate buffer, pH 6.5. The intensity of the red-colored bands, developed directly on the membrane, was quantified using a computing, laser densitometer and shown to be linearly proportional to the original enzyme activity in extracts determined by liquid assay. The temperature inactivation profile of wax moth trehalase was measured. Alteration of the electrophoresis sample buffer composition further revealed the presence of putative trehalase isoforms in wax moth larval extracts whose relative levels of activity were altered during the course of starvation and infection with Tipula iridescent virus.

Animals↗
PubMed1983

Trehalase from the cellular slime mold Dictyostelium discoideum: purification and characterization of the homogeneous enzyme from myxamoebae.

Trehalase (alpha-alpha'-trehalose 1-D-glucohydrolase, EC 3.2.1.28) was solubilized from myxamoebae of the cellular slime mold Dictyostelium discoideum by a freeze-thaw cycle and was subsequently purified to homogeneity using the techniques of ethanol fractionation, molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide disc gel electrophoresis. The 1000-fold purified enzyme had a specific activity of about 104 units/mg, which was accompanied by a net recovery of 5 to 7% of the original activity. The purified enzyme was maximally active at pH 5.5, showed high specificity for trehalose, and exhibited a typical hyperbolic response as a function of trehalose concentration with a Km of 1.2 mM. The enzyme was maximally active at 50 degrees C and had an energy of activation of 12-13 kcal/mol. Thermal stability studies demonstrated that full enzymatic activity was recovered following a 5-min incubation of trehalase at temperatures up to 45-50 degrees C. Analysis of various compounds for inhibitory effects indicated that Tris and urea were slightly effective, reducing enzymatic activity by 28 and 6% at concentrations of 100 and 10 mM, respectively. Of five heavy metals tested, HgCl2 was the most inhibitory, reducing activity by 58% when present at a final concentration of 1.0 mM. Enzymatic activity was not affected by any adenine derivative examined (e.g., ATP, ADP, AMP, cAMP, adenosine, and adenine). The molecular weight of the native enzyme was determined by molecular sieve chromatography, pore gradient electrophoresis, and electrophoresis as a function of acrylamide concentration. All three methods yielded a value of about 10(5) +/- 5 X 10(3). Estimation of the subunit or monomer molecular weight by sodium dodecyl sulfate-gel electrophoresis indicated a value of 95-100 X 10(3). The isoelectric point as determined in 7.5% polyacrylamide gels with pH 3-10 ampholytes was 7.2-7.3. The purified enzyme adsorbed to concanavalin A-Sepharose in the presence of KCl (0.1 M) and was eluted with alpha-methylmannoside, thereby suggesting an association between trehalase and carbohydrate. In agreement with this conclusion was the observation that trehalase could be specifically stained for carbohydrate with the Alcian blue and periodic acid-Schiff's reagents following polyacrylamide disc gel electrophoresis.

Amino Acids↗