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PubMed1973

Activity and heat stability of trehalase from the mycelium and ascospores of Neurospora.

Trehalases from the ascospores of Neurospora tetrasperma and the mycelium of N. crassa were compared. Enzymes from both sources have identical electrophoretic mobilities, K(m)'s, responses to pH, immunological reactions, and activities in low-molarity buffers. Because both enzymes are so similar, conclusions about the properties of the ascospore enzyme may, be made by studying mycelial trehalase. Mycelial trehalase is most active and stable in low-molarity buffers. The enzyme exists in at least three species; the smallest has a molecular weight between 105,000 and 125,000 and is predominant in low-molarity buffers at 37 C. The stability of trehalase to heating at 65 C can be increased by increasing enzyme concentration and by the addition of polyols. Ascospores contain large amounts of trehalose, which protects trehalase from heat inactivation at 65 C. The importance of this phenomenon in vivo and its relationship to the localization of trehalase in ascospores is discussed.

Antigens, Fungal↗
PubMed1984

Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores.

Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity.

Adenosine Triphosphate↗
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↗
PubMed1987

Trehalase of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions.

Escherichia coli can use the nonreducing disaccharide trehalose as a sole source of carbon and energy. Trehalose transport into the cell is mediated via the phosphotransferase system, and a mutant depleted in the nonspecific proteins enzyme I, HPr, and enzyme IIIGlc of this system was not only unable to grow on glucose or mannitol but also was strongly reduced in its ability to grow on trehalose. A pseudorevertant (PPA69) of such a deletion mutant was isolated that could again grow on glucose but not on mannitol. This revertant could now also use trehalose as a carbon source due to a constitutive galactose permease. PPA69 was subjected to Tn10 insertional mutagenesis, and a mutant (UE5) was isolated that no longer could use trehalose as a carbon source but could still grow on glucose. UE5 lacked a periplasmic trehalase that was present in PPA69. P1-mediated transduction of this Tn10 insertion (treA::Tn10) into a pts+ wild-type strain (MC4100) had no effect on the ability of MC4100 to grow on trehalose but resulted in loss of the periplasmic trehalase activity. The Tn10 insertion was mapped at 26 min on the E. coli linkage map and was 3% cotransducible with trp, in the order treA::Tn10, trp, cys. Trehalase activity in MC4100 was not induced by growth in the presence of trehalose but increased by about 10-fold when 0.6 M sucrose was added to minimal growth medium. Using the in vivo mini-Mu cloning system and growth on trehalose as selection, we cloned the treA gene. A 9-kilobase EcoRI fragment containing treA was subcloned into pBR322. Strains carrying this plasmid (pTRE5) contained about 100-fold higher periplasmic trehalase activity than PPA69 or MC4100. Using polyacrylamide gel electrophoresis, we found a protein of molecular weight 58,000 among the periplasmic proteins of the pTRE5-carrying strain that was absent in UE5. This protein was purified by ammonium sulfate precipitation and DEAE-Sepharose ion-exchange chromatography and contained all the trehalase activity. Minicells containing the treA+ plasmid produced, in addition to three other proteins, the 58,000-dalton protein. Thus, the plasmid carries the structural gene for the periplasmic trehalase and not just a gene involved in the regulation of the enzyme.

Cloning, Molecular↗
PubMed1988

Purification and characterization of acid trehalase from the yeast suc2 mutant.

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.

Amino Acids↗
PubMed1993

Assay of trehalose with acid trehalase purified from Saccharomyces cerevisiae.

An enzymatic end-point assay of trehalose using acid trehalase from yeast is described. After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system. Pre-existing glucose is determined in a control reaction from which acid trehalase is omitted. When intact cells are analysed for trehalose, pre-existing glucose can be washed out with ice-cold water without reducing the trehalose content of the cells. A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95 degrees C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid trehalase preparation used. As described previously (Mittenbühler, K. and Holzer, H., 1988, J. Biol. Chem. 263, 8537-8543; Mittenbühler, K., 1988, Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose-6-phosphate, glucose-1-phosphate, galactose. The application of the new trehalose assay to yeast cells grown to different growth stages and at various temperatures is presented.

Biological Assay↗
PubMed1984

Cyclic-AMP content and trehalase activation in vegetative cells and ascospores of yeast.

Addition of glucose to yeast ascospores, glucose-grown vegetative cells from the stationary growth-phase or acetate-grown vegetative cells from the logarithmic growth-phase induces a rapid tenfold increase in the activity of trehalase. Trehalase activation is followed by a period of slow inactivation. It was possible to reverse the inactivation in the presence of glucose in all cell types immediately and completely by subsequent addition of a nitrogen source. This reactivation by nitrogen sources is in disagreement with proteolytic breakdown being responsible for trehalase inactivation in the presence of glucose. The addition of glucose induced in all cell types a rapid transient increase of the cellular cyclic-AMP content. In ascospores the increase of the cyclic-AMP level was about twofold, in glucose-grown stationary-phase vegetative cells four- to fivefold and in acetate-grown vegetative cells about sevenfold. Subsequent addition in the presence of glucose of a nitrogen source caused a new twofold increase of the cyclic-AMP level in ascospores. In the other two cell types however addition of a nitrogen source after the initial transient increase of the cyclic-AMP level did not produce a significant new increase. Although the data obtained for ascospores at first seemed to confirm the crucial role of the increase in the cyclic-AMP level for the activation of trehalase, the data obtained afterwards for vegetative cells indicated that it is possible to activate trehalase in yeast without a concomitant increase of the total cellular cyclic-AMP content.

Cyclic AMP↗
PubMed2004

Nucleotide sequence variation does not relate to differences in kinetic properties of neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

Genetic variability in a putative virulence factor, the neutral trehalase ( Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., Km, Vmax, and Kcat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower Kcat/Km. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.

Amino Acid Substitution↗
PubMed1987

Purification and properties of detergent-solubilized pig kidney trehalase.

Trehalase (alpha, alpha-trehalase, EC 3.2.1.28) was solubilized from the brush border membrane of pig kidney cortex by Triton X-100 and sodium deoxycholate in the presence of inhibitors of proteolytic enzymes. The kidney enzyme was purified 3060-fold using gel filtration, ion exchange chromatography, Con A-Sepharose chromatography, phenyl-Sepharose CL-4B hydrophobic interaction chromatography, Tris-Sepharose 6B affinity chromatography, and hydroxylapatite chromatography. Tris-Sepharose 6B was utilized to absorb contaminant proteins. Purity was estimated as 99% or greater, based on amino-terminal amino acid analysis. The purified enzyme had a specific activity of 278 units/mg protein, showed one major band after silver staining, and had an estimated molecular weight of 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was a glycoprotein and contained 2 mol of glucosamine per mole of trehalase. Kidney trehalase was inhibited by Tris, HgCl2, and phlorizin with Ki values of 3.8 mM, 11 microM, and 2.4 mM, respectively. Inclusion of Cl- in the reaction mixture protected the enzyme from inactivation by HgCl2. The apparent Km for trehalose was calculated to be 2.1 mM. Kidney trehalase was highly specific for trehalose and exhibited an optimal pH of 5.9. The isoelectric point was between pH 4.7 and 4.4, as measured by chromatofocusing.

Animals↗
PubMed1982

Does a cyclic AMP-dependent phosphorylation initiate the transfer of trehalase from the cytosol into the vacuoles in Saccharomyces cerevisiae?

Trehalase activity in a yeast protoplast lysate increased approximately 40-times upon preincubation with cAMP and ATP. The activity present without the preincubation could all be sedimented at 8000 x g, for 10 min confirming the previously reported localization of the active trehalase (Ta) in the vacuoles. Virtually all the trehalase activity newly formed upon the preincubation, however, was found in the soluble fraction, indicating that a trehalase-zymogen (Tz) is located in the cytosol. This raises the possibility that a cAMP-dependent phosphorylation not only transforms Tz to Ta but also initiates the transfer of trehalase from the cytosol into the vacuoles.

Cyclic AMP↗
PubMed1995

Activation of neutral trehalase by glucose and nitrogen source in Schizosaccharomyces pombe strains deficient in cAMP-dependent protein kinase activity.

Schizosaccharomyces pombe cells carrying a disruption in the PKA1 gene, that encodes the catalytic subunit of cAMP-dependent protein kinase (PKA), lacked the glucose- and nitrogen-source-induced activation of trehalase at stationary-phase but rised trehalase activity in response to these compounds during the exponential phase of growth. Treatment by phosphatase of either glucose- or nitrogen-source-activated trehalase resulted in trehalase deactivation suggesting that phosphorylation of the enzyme protein occurs during activation. These data indicate that in growing cells of this yeast the mechanism responsible for the activation of trehalase can be independent of interactions with free catalytic subunits of PKA and related to a signaling pathway involving a type of protein kinase different from PKA.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1983

Inhibition of an insect midgut trehalase by dioxane and delta-gluconolactone: enzyme pKa values and geometric relationships at the active site.

1. Inhibition of the Rhynchosciara americana midgut trehalase (alpha, alpha'-trehalose glucohydrolase, EC 3.2.1.28) by the competitive inhibitor dioxane have been studied. 2. Determinations of the Ki of dioxane at different pH provided the true pKa values of the prototropic groups of the trehalase active site (pKa 5.2 and 8.2), which are in agreement with those determined previously from kinetic and chemical modification data. 3. Dioxane only changes the enzyme pKa values if bound at the enzyme active site. 4. Gluconic acid does not inhibit the trehalase, in contrast to delta-gluconolactone which is a simple intersecting linear inhibitor. 5. Multiple inhibition analyses showed that delta-gluconolactone and Tris bind at the same site in the trehalase active center, whereas dioxane binds closer to delta-gluconolactone than to Tris. 6. The data support the assumption that dioxane binds at the middle portion of the trehalase active site.

Animals↗
PubMed2002

Developmental expression of trehalase: role of transcriptional activation.

The third postnatal week of mouse development is characterized by dramatic changes of gene expression in the small intestine. Although these changes are often assumed to reflect regulation at the level of transcription, to date there have been no direct investigations of this. In the current study we have used trehalase as a marker of intestinal maturation. Highly sensitive reverse transcriptase-polymerase chain reaction methods were developed for semi-quantitative analysis of both initial and mature transcripts, i.e., hnRNA and mRNA. Jejunums collected during normal development (specifically from postnatal days 8-21) showed parallel increases in the levels of trehalase hnRNA and mRNA. Likewise, when precocious gut maturation was elicited by dexamethasone administration on days 8-10, both initial and mature trehalase transcripts were significantly increased, although with a relatively slow time course. We conclude that both normal and glucocorticoid-induced maturation of trehalase expression reflect transcriptional activation. However, the slow time course of the glucocorticoid effect suggests that trehalase may not be a primary response gene.

Age Factors↗
PubMed1997

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose.

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.

Amino Acid Sequence↗
PubMed2004

The ATC1 gene encodes a cell wall-linked acid trehalase required for growth on trehalose in Candida albicans.

After screening a Candida albicans genome data base, the product of an open reading frame (IPF 19760/CA2574) with 41% identity to Saccharomyces cerevisiae vacuolar acid trehalase (Ath1p) was identified and named Atc1p. The deduced amino acid sequence shows that Atc1p contains an N-terminal hydrophobic signal peptide and 20 potential sites for N-glycosylation. C. albicans homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two ATC chromosomal alleles. Analysis of these null mutants shows that Atc1p is localized in the cell wall and is required for growth on trehalose as a carbon source. An Atc1p endowed with acid trehalase activity was obtained by an in vtro transcription-translation coupled system. These results strongly suggest that ATC1 is the structural gene encoding cell wall acid trehalase in C. albicans. Determinations of ATC1 mRNA expression as well as acid trehalase activity in the presence and absence of glucose point out that ATC1 gene is regulated by glucose repression.

Alleles↗
PubMed1981

Methods for the rapid purification of trehalase from Dictyostelium discoideum.

A rapid and reliable method for the preparation of homogeneous trehalase from the cellular slime mold, Dictyostelium discoideum for usage in enzyme characterization studies and trehalose assays was developed. This procedure takes advantage of the fact that trehalase activity is secreted by Dictyostelium during the course of development, the major fraction being released late in fruiting body formation. Purification of trehalase to electrophoretic homogeneity was accomplished utilizing the techniques of ultrafiltration, streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel chromatography and preparative disc gel electrophoresis. Analysis of the purified enzyme by analytical polyacrylamide disc gel electrophoresis demonstrated the presence of a single protein band which was stainable with Coomassie blue. Assay of trehalase activity in eluates from segments of a companion gel indicated that all of the recovered trehalase activity was associated with this band of protein. Examination of the substrate specificity of the purified enzyme indicated absolute specificity for trehalose.

Dictyostelium↗
PubMed2004

Regulation of intestinal ontogeny: effect of glucocorticoids and luminal microbes on galactosyltransferase and trehalase induction in mice.

Intestinal maturation can be influenced by intrinsic factors (glucocorticoid hormones) and by extrinsic factors (resident microflora); their relative roles in ontogeny of mouse intestinal trehalase expression, a marker of general gut development, and of beta1,4-galactosyltransferase (beta GT), a marker of glycosyltransferase development, were investigated. In conventional (CONV) mice, beta GT and trehalase gene expression rapidly increased to adult levels by the fourth postnatal week. In germ-free (GF) mice, beta GT expression remained at initial low levels and was rapidly induced on reintroduction of luminal microbes of the adult gut but not of microbes characteristic of the suckling gut. Similar developmental patterns were observed for colonic galactosyl beta1,4-linked glycoconjugates, products of beta GT activity. These results indicate an essential role for microbes in the ontogeny of beta GT. In both CONV and GF mice, cartisone acetate (CA) precociously accelerated the ontogeny of beta GT and trehalase until maturation of the gut occurred (day 22). In the mature gut of CONV mice, both beta GT and trehalase are elevated and insensitive to CA; in GF mature mice, the expression of beta GT remains low, whereas the expression of trehalase was at mature levels, regardless of CA treatment. These changes in enzyme activity were accompanied by parallel changes in mRNA, implying transcriptional regulation. Thus both microbes and cortisone regulate gut ontogeny, but only suckling gut responds to CA, an intrinsic factor, whereas adult gut beta GT expression remains sensitive to microflora, an extrinsic factor. However, induction of the adult pattern of glycosyltransferase expression in mature gut requires colonization by microflora typical of adult gut, suggesting an essential role for intestinal colonization in the ontogeny of normal intestinal mucosal cell surface glycoconjugate receptors.

Animals↗
PubMed2006

Identification of an extracellular acid trehalase and its gene involved in fungal pathogenesis of Metarizium anisopliae.

Trehalose is the main sugar in the haemolymph of insects and is a key nutrient source for an insect pathogenic fungus. Secretion of trehalose-hydrolysing enzymes may be a prerequisite for successful exploitation of this resource by the pathogen. An acid trehalase [EC 3.2.1.28] was purified to homogeneity from a culture of a locust-specific pathogen, Metarhizium anisopliae, and its properties were characterized. The gene (ATM1) of this acid trehalase was also isolated. The pure enzyme can efficiently hydrolyze haemolymph trehalose into glucose in vitro. The new acid trehalase appearing in the haemolymph of Locusta migratoria infected with M. anisopliae had the same pI and substrate specificity as the purified fungal acid trehalase, and the concentration of trehalose in the haemolymph decreased sharply after infection. RT-PCR also revealed the ATM1 gene's expression in the haemolymph of the infected insects. Our results indicated that the acid trehalase may serve as an "energy scavenger" and deplete blood trehalose during fungal pathogenesis.

Amino Acid Sequence↗