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At least 19 recordsLinked to original sources

PubMed1991

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase.

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.

Cell Wall↗

PubMed1990

Rabbit small intestinal trehalase. Purification, cDNA cloning, expression, and verification of glycosylphosphatidylinositol anchoring.

alpha,alpha-Trehalase (EC 3.2.1.28), an intrinsic protein of intestinal brush-border membranes, was purified to homogeneity from rabbits. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequence of a CNBr peptide were employed in a polymerase chain reaction to amplify a 71-base pair fragment of trehalase DNA with rabbit intestine cDNA as a starting template. This fragment was used as a hybridization probe to isolate full length trehalase clones from a rabbit intestine cDNA bank. Sequence analysis revealed that trehalase comprises 578 amino acids, contains at the amino terminus a typical cleavable signal sequence, at the carboxyl terminus a rather hydrophobic region typical of proteins anchored via glycosylphosphatidylinositol, and four potential N-glycosylation sites. Trehalase has no sequence homologies with other sequenced brush-border glycosidases. Northern blot analysis revealed a 1.9-kilobase trehalase mRNA in small intestine and kidney, smaller amounts in liver, and none in lung. Southern blot analysis indicated the gene has a length of 20 kilobase pairs or less. Injection into Xenopus laevis oocytes of mRNA synthesized in vitro from a trehalase template resulted in the expression of trehalase activity several hundredfold above background. The trehalase activity was membrane-bound and could be solubilized upon digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. This strongly suggests that rabbit small intestinal trehalase is anchored via glycosylphosphatidylinositol also when expressed in X. laevis oocytes.

Amino Acid Sequence↗

PubMed1975

Properties of a free and a solubilized form of bound alpha,alpha-trehalase purified from honey bee thorax.

The free and bound forms of alpha,alpha-trehalase (EC 3.2.1.28) of the honey bee thorax were separated and the bound enzyme was solubilized by raising the pH to 8.0 for 10 h. Both enzymes were purified. They were homogeneous as determined by several electrophoretic criteria. It was found that the two enzymes had very similar Km's (each about 0.89 mM), Vm's (53.2 and 54.3 U/mg for free and solubilized, respectively), inhibition characteristics, specificities (both only hydrolyzed alpha,alpha-trehalose), pH maxima (each had maxima at about 3.5 and 6.5), molecular weights (65,000), isoelectric points (5.1), reactivities to sulfhydryl reagents, electrophoretic mobilities, activation energies (about 12.8 kcal/mol), and similar stabilities to heat, pH, and urea. Some significant differences between the two enzymes were, however, found: the solubilized alpha,alpha-trehalase floated at 70% saturation of ammonium sulfate while the free alpha,alpha-trehalase did not; the solubilized alpha,alpha-trehalase did not dissociate into subunits as readily as did the free one; and the solubilized alpha,alpha-trehalase was found to bind more readily to a hydrophobic grouping than the free enzyme. In addition to these comparisons, three new findings relating to thorax alpha,alpha-trehalases are reported. (1) Thorax alpha,alpha-trehalases are strongly inhibited by beta-glucosides (Ki values of about 8 x 10(-4) M); (2) under certain conditions thorax alpha,alpha-trehalases from honey bees dissociated into subunits of one-half the normal molecular weight; (3) honey bee thorax alpha,alpha-trehalases have unusual biphasic pH activity profiles.

Animals↗

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↗

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↗

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↗

PubMed1979

The kinetic parameters of trehalase in whole and disrupted mitochondrial preparations from two insects with asynchronous muscle.

The kinetic parameters of trehalase in honey bee and flesh fly mitochondria were compared. The studies were carried out with whole mitochondria and with mitochondria disrupted in various ways and to different degrees. Honey bee mitochondrial trehalase was significantly activated by Lubrol WX treatment (30.0-fold), by high pH treatment (20.8-fold), and by a treatment consisting of 10 passes through a French press (37.9-fold) but not by the other treatments tried (salt, proteases, Waring blender, and sonication), despite the fact that these treatments also disrupted the mitochondria significantly. The activation effect was on the Vmax. The Km value did not change. Simple breakage of either the outer or inner (or both) membranes was not sufficient to activate trehalase from honey bees, which showed that the activation was not an indirect result of a change in the case with which trehalose can pass through the membranes. Honey bee trehalase is the first trehalase from insects with asynchronous muscle which has been shown to be activatable by physical and chemical methods. Flesh fly mitochondrial trehalase behaved quite differently from the honey bee enzyme in that it could not be activated by any of the techniques tried, even when there were significant amounts of disruption.

Animals↗

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↗

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↗

PubMed1991

A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast.

Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods. The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0-4 degrees C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30 degrees C. The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (less than 1 min) activation during handling at 4 degrees C before homogenization. Activity of the enzyme in these homogenates decreased 75-90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation. The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27 degrees C, and very low activities again for cells shifted from 27 to 40 degrees C for a second time. Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S. cerevisiae by phosphorylation and dephosphorylation.

Cell Membrane Permeability↗

PubMed1991

Activation of yeast trehalase by heat shock.

1. Activation of Saccharomyces cerevisiae trehalase by heat shock was shown in all strains tested, including mutants in which the response to a glucose signal was absent. A low concentration of cAMP favored the response as seen in 2nd log cells or in ras2 and cyr1ts mutant strains. The heat shock effect upon trehalase activity was not observed under conditions of catabolite repression. 2. Neither hexokinase PII nor the heat shock protein hsp26 seemed to be involved in the activation of trehalase by heat shock. However, mutant strains deleted in the polyubiquitin gene showed only a 2-fold activation of the enzyme while in control strains a 5- to 7-fold irreversible activation was observed. 3. An alternative mechanism of trehalase activation by removal of an inhibitor through ligation with ubiquitin is discussed. Activation by cAMP-independent phosphorylation is also considered.

Culture Media↗

PubMed1991

Modulation of trehalase activity in two insect cell lines by virus infection and trehalose.

Trehalase (EC 3.2.1.28), an important glycosidase involved in regulating trehalose levels and metabolic energy in insects, was measured in cell lines from fall army worm, Spodoptera frugiperda and salt marsh caterpillar, Estigmene acrea, treated with either glucose or trehalose in the presence or absence of Tipula Iridescent Virus (TIV), a cytoplasmic deoxyribovirus. In medium containing 15-35 mM trehalose, both of these cells increased their trehalase activities by 4.5 to 8x the basal levels from cells in glucose medium. Trehalase activity was rapidly reduced after cells were exposed to TIV. Maximum loss in activity (70-90%), occurring about the same time as peak viral DNA synthesis, was significantly delayed when cells were pre-incubated with 30 mM trehalose. These experiments demonstrate the potential utility of trehalase as a marker for monitoring stresses induced by viral infection and changes in nutrition.

Animals↗

PubMed1979

Effects of cortisone and thyroxine on intestinal trehalase activity in infant mouse.

Cortisone acetate (25 microgram/g b.wt/day) administration to 8-day-old suckling mice induces a premature increase of trehalase activity along the entire small intestine. On the other hand, thyroxine (1 microgram/g b.wt/day) in unable to provoke a precocious increase of trehalase activity. Trehalase appears to be the only brush border membrane disaccharidase controlled solely by glucocorticoid hormones during the postnatal maturation of the intestine.

Animals↗

PubMed1978

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa.

Trehalase (alpha,alpha-Trehalose glucohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50 degrees C in acetate buffer at pH 5.5. It was highly specific for alpha,alpha-trehalose and had an apparent Km = 0.4 mM at 50 degrees C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170 000. Trehalase from mycelium grown at 40 and 50 degrees C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by "protection against heat-inactivation by "protective factor(s)" present in mycelial extracts. The "protective factor(s)" was sensitive to proteolytic digestion. It was not diffusible and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.

Dialysis↗

PubMed1975

Histochemical localization of trehalase activity in dorsal flight muscle of the flesh fly Sarcophaga bullata with light and electron microscopy.

Trehalase activity in flight muscle of the flesh fly Sacrophaga bullata is detected histochemically at light- and electron-microscopic levels by using diaminobenzidine, glucose oxidase and peroxidase in the incubation medium. The association of trehalase activity with the inner mitochondrial membrane is confirmed. Biochemical assay shows that about 50% of the initial total trehalase activity is lost from the tissue during the histochemical processing and about 50% remains for histochemical detection.

Animals↗

PubMed1975

[Influence of parenterally injected trehalose in mammals having trehalase activity at different sites].

When trehalose is injected via parenteral pathway into animals lacking kidney trehalase (rat), more than 75 per cent of this disaccharide is eliminated in urine. When the injected animals possess an active kidney trehalase (guinea-pig, rabbit), there is only a low urinary trehalose excretion. Moreover, in rabbit, a marked hyperglycaemia is observed which is due to the rapid hydrolysis of trehalose by kidney trehalase.

Animals↗

PubMed1992

Trehalase activity and its regulation during growth of Saccharomyces cerevisiae.

Trehalase activity decreased in 95% at the onset of the transition phase of growth of S. cerevisiae. The question which we raised was whether this phenomenon was due to proteolysis or to conversion of the enzyme to a less active form (dephosphorylation). Immunological methods allowed to identify the presence of the trehalase protein during cell growth. At the same stage of growth, an increase in the non-phosphorylated enzyme was detected "in vitro". Results utilizing mutant strains also indicated that regulation occurred by interconversion of forms. The same mechanism also seems to control trehalase activity in non proliferating conditions.

Blotting, Western↗

PubMed1977

Trehalase activity in diabetes mellitus and in cirrhosis of the liver.

Trehalase (an enzyme decomposing the disaccharide trehalose) activity was studied in 29 healthy subjects, 25 patients with cirrhosis and 112 diabetics. Mean trehalase activity was 176 +/- 11 units in the control group, 647 +/- 421 units in the patients with cirrhosis and 467 +/- 239 units in diabetics. The differences between the control group on the one hand and the groups with cirrhosis and diabetes on the other were statistically significant. The results show that the organism, under pathological conditions, makes far greater use of its enzymatic apparatus to assure its basic requirements, but the scatter of the values is so great that the determination of trehalase has no discriminative value in individual cases.

Aged↗