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PubMed1989

Steric course of the hydrolysis of alpha,alpha-trehalose and alpha-D-glucosyl fluoride catalyzed by pig kidney trehalase.

We are unable to confirm the report of Labat et al.3 that pig kidney trehalase hydrolyzes alpha,alpha-trehalose to form solely alpha-D-glucose. Highly purified trehalase from pig renal cortex was found, in reactions monitored by 1H-n.m.r. spectra, to hydrolyze alpha,alpha-trehalose with the formation of both alpha- and beta-D-glucose. That the beta anomer constitutes the enzymically mobilized glucosyl residue is indicated by the further finding that beta-D-glucose is the product formed on hydrolysis of alpha-D-glucosyl fluoride by the enzyme. Present results show the stereochemical behavior of pig kidney trehalase in hydrolyzing alpha,alpha-trehalose to be indistinguishable from that reported by ourselves and others for trehalase preparations from a range of biological sources including rabbit renal cortex.

Animals↗
PubMed1986

Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C.

Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic. Phosphatidylinositol-specific phospholipase C also converted Triton X-100-solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.

Alkaline Phosphatase↗
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↗
PubMed1984

Trehalases from the male accessory glands of the American Cockroach: developmental changes and the hormonal regulation of the enzymes.

Two types of trehalases, designated CM-I and CM-II, were detected in the male accessory gland of the American cockroach and they could be separated by CM-cellulose chromatography (S. Y. Takahashi, S. Higashi, S. Minoshima, M. Ogiso, and K. Hanaoka, 1980, Int. J. Invert. Reprod. 2, 373-381). Trehalase activity in the gland showed a rapid increase after adult emergence. The relative activities of the two enzymes were followed separately during adult development. The appearance of CM-II preceded that of CM-I during adult development. Allatectomy and decapitation of newly molted adults resulted in inhibition of the increase of enzyme activity, and in the allatectomized cockroach, CM-II, which is the major enzyme activity in the gland, was missing. Implantation of the corpora allata as well as application of JH-III and the JH analogs isopropyl-11-methoxy-3,7,11-trimethyl-2,4,-dodecadienoate (ZR-515) and 6,7-epoxy-1-(p-ethylphenoxy)-3,7-dimethyl-2-octene (R-20458) into the decapitated animals restored the enzyme activities. The data suggested that trehalase in the male accessory gland was under the control of the corpora allata. The regulation of trehalase activity in the male accessory gland was discussed with respect to its function.

Animals↗
PubMed1993

Purification, cDNA cloning and northern blot analysis of trehalase of pupal midgut of the silkworm, Bombyx mori.

Trehalase (alpha-glucoside-1-glucohydrolase, EC 3.2.1.28) was purified from silkworm pupal midgut to homogeneity by DEAE-Sepharose CL-6B and hydroxyapatite chromatography, and native gel electrophoresis. The enzyme had a molecular mass of 70 kDa. The N-terminal amino-acid sequence of the intact trehalase and its three fragments by V8 proteinase digestion was determined. Based on the amino-acid sequence, degenerate oligonucleotides were synthesized and used as primers in a polymerase chain reaction (PCR). Using a 0.8 kb PCR product as a hybridization probe, trehalase clones were isolated from the pupal midgut cDNA library. Sequence analysis revealed that the isolated trehalase cDNA contains 3103 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis clearly showed a 3.0 kb transcript in midgut, and Malpighian tubule, but not in fat body, silk gland, ovary, trachea, brain and suboesophageal ganglion.

Amino Acid Sequence↗
PubMed1982

Effect of inorganic anions of the inhibition of trehalase activity by mercuric chloride.

Monovalent inorganic anions showed an unexpected effect on the inhibition of trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) by SH inhibitors. This phenomeon (deinhibition) was caused by monovalent anions, Cl-, Br-, I- and SCN- . F- and ClO4- showed partial deinhibition. Deinhibition was not caused by NO2- and SO4-. The effectiveness of the "active anions' in causing deinhibition was highly dependent on the anion size. Trehalase in the presence of mercuric chloride was "activated' by Cl-, and the activation was saturable. From the results of Dixon plots for trehalase at different concentrations of the "activator' (deinhibitor) and a constant concentration of the substrate, it can be seen that the activator and the inhibitor competed with each other. Thus, it is suggested that the activator and the inhibitor share a common binding site or bind very near each other. The Ki value for mercuric chloride was increased with increasing concentration of NaCl. Therefore, it might be essential to remove the "active anions' in order to determine the inhibitory effect and the Ki value of trehalase for SH inhibitors.

Animals↗
PubMed1986

Maltase-glucoamylase and trehalase in the rabbit small intestine and kidney brush border membranes during postnatal development, the effects of hydrocortisone.

Kidney and intestinal brush border membranes were isolated from 14-day-old rabbits and papaïn solubilized maltase-glucoamylase was purified to almost homogeneity from both membranes. Maltase-glucoamylase from kidney and intestine have the same molecular weight (669,000 daltons by AcA 22 gel filtration) and the same Km (4 mM, for maltose). Tris (Ki = 12.5 mM, for maltose) is a non-competitive inhibitor for both enzymes. In intestine, maltase and glucoamylase have low activity during the first two postnatal weeks and then undergo a sharp increase during the next 2 weeks. In contrast, for trehalase, adult levels are reached about 6 days after birth. Hydrocortisone injection to 10 days rabbits causes precocious increases in the specific activities of trehalase (3.6 x), maltase (5.2 x) and glucoamylase (7.4 x). Conversely, kidney maltase, glucoamylase and trehalase activities rise gradually from birth, reaching adult levels by the end of the third week. Administration of hydrocortisone to suckling rabbit does not affect either trehalase or maltase and glucoamylase in kidney brush border membrane.

Aging↗
PubMed1984

Influence of octopamine on trehalase activity in muscle and hemolymph of the American cockroach, Periplaneta americana L.

Injection of adult male cockroaches (Periplaneta americana) with 10 microliter 1 microM octopamine causes elevated activity of trehalase (alpha, alpha-trehalose glucohydrolase; EC 3.2.1.28) in hemolymph and muscle but not in gut. Tyramine, dopamine and glutamate, at the same concentration, failed to elicit any effect on trehalase activity. Determination of some kinetic parameters for muscle and hemolymph trehalases reveal that octopamine causes an increase in Vmax without any significant alteration in the Km of the enzyme for trehalose. The results are discussed in terms of the physiological significance of octopamine-mediated activation of tissue trehalases.

Animals↗
PubMed1995

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger.

Resting cells of the fission yeast Schizosaccharomyces pombe, suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen tannophilus, the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.

Asparagine↗
PubMed2002

Molecular cloning, characterisation, and expression of a neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

A neutral trehalase gene (NTH1) was isolated from a lambdaEMBL3 genomic library of the insect pathogenic fungus Metarhizium anisopliae. Sequencing of the gene revealed extensive homology with other fungal neutral trehalases. The NTH1 gene exists as a single copy in the genome. Two STREs exist in the 5'UTR of NTH1, which may mediate transcriptional activation of the NTH1 gene in response to various stresses. The NTH1 gene encodes a protein of 737 amino acids with a calculated M(r) of 83.1kDa. A cyclic adenosine 3',5'-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site were found in the amino-terminal domain of NTH1, consistent with a regulatory enzyme. Expression of the trehalase cDNA was achieved in Saccharomyces cerevisiae. Southern blot analysis of RT-PCR products indicated that the neutral trehalase gene is transcribed in vitro in cell-free haemolymph of the tobacco hornworm Manduca sexta and in vivo in the early stage of infection.

Animals↗
PubMed1997

Modulation of trehalase activity in Saccharomyces cerevisiae by an intrinsic protein.

The regulation of cytosolic trehalase activity in yeast has been described as cycles of activation by phosphorylation by cAMP protein kinase. In this paper, evidence is presented for another regulatory mechanism--the binding of an endogenous inhibitory protein. This negative modulator was isolated during the purification procedure of cytosolic cryptic trehalase from repressed wild-type cells of Saccharomyces cerevisiae. However, in derepressed cells the inhibitor was not found nor was it present in ras2 mutant cells submitted to a heat treatment. The trehalase inhibitory activity proved to be a calmodulin ligand protein and, therefore, involved in the modulation of trehalase activity by Ca2+ ions.

Fungal Proteins↗
PubMed1998

Comparative studies of suidatrestin, a specific inhibitor of trehalases.

Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor. Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases. The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast. Suidatrestin was inactive on maltase but inhibited all trehalases with IC50 values of 0.08-0.1 microM; Ki values ranged from 0.02 to 0.05 microM. The very low Ki/K(m) ratios (3.9 x 10(-6) -4.9 x 10(-6)) indicated excellent in vitro inhibitory action of suidatrestin. When injected into larvae of S. littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only. Consecutive omission of the inhibitor even stimulated weight increase above that of controls. Significant mortality was achieved at a rather high dose only. Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration. The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.

Animals↗
PubMed1997

Molecular cloning, sequencing and expression of cDNA encoding human trehalase.

A complete cDNA clone encoding human trehalase, a glycoprotein of brush-border membranes, has been isolated from a human kidney library. The cDNA encodes a protein of 583 amino acids with a calculated molecular weight of 66,595. Human enzyme contains a typical cleavable signal peptide at amino terminus, five potential glycosylation sites, and a hydrophobic region at carboxyl terminus where the protein is anchored to plasma membranes via glycosylphosphatidylinositol. The deduced amino acid sequence of the human enzyme showed similarity to sequences of the enzyme from rabbit, silk worm, Tenebrio molitor, Escherichia coli and yeast. Northern blots revealed that human trehalase mRNA of approx. 2.0 kb was found mainly in the kidney, liver and small intestine. Expression of the recombinant trehalase in E. coli provided a high level of the enzyme activity. The isolation and expression of cDNA for human trehalase should facilitate studies of the structure of the gene, as well as a basis for a better understanding of the catalytic mechanism.

Amino Acid Sequence↗
PubMed2001

Synthesis and quantitative structure-activity relationships of fluorine-containing 4,4-dihydroxylmethyl-2-aryliminooxazo(thiazo)lidines as trehalase inhibitors.

Five fluorine-containing 4,4-dihydroxylmethyl-2-aryliminooxazolidines and five 4,4-dihydroxylmethyl-2-aryliminothiazolidines were synthesized and evaluated for their inhibitory activity against trehalase in vitro. All these compounds were very readily synthesized compared with the natural trehalase inhibitors. They had moderate inhibitory activity toward trehalase, and showed larvicidal activity and inhibition action to insect flight. The steric parameters and semiempirical quantum parameters of these compounds were acquired by using the molecular modeling method and the PM3-SCF-MO method, respectively. A quantitative structure-activity relationship between half-inhibitory concentrations toward trehalase and the above parameters was established.

Animals↗
PubMed2003

A role for calcium in the regulation of neutral trehalase activity in the fission yeast Schizosaccharomyces pombe.

Neutral trehalases mobilize trehalose accumulated by fungal cells as a protective and storage carbohydrate. A structural feature of these enzymes is the presence of an EF-like motif similar to that shown by many Ca2+-binding proteins. In this study we provide direct evidence for physical binding of Ca2+ to neutral trehalase (Ntp1p) of the fission yeast Schizosaccharomyces pombe, and show that aspartic residues at positions 97 and 108 in the conserved putative Ca2+-binding motif of Ntp1p appear to be responsible for this interaction. Mutations in these residues do not interfere with the ability of Ntp1p to associate in vivo with trehalose-6-phosphate synthase, but prevent activation of neutral trehalase triggered by the addition of glucose or by subjecting cells to stressing conditions. Strains expressing Ntp1p variants that are unable to bind Ca2+ partially resemble those devoid of the ntp1+ gene in terms of trehalose hyperaccumulation. Gel filtration of cell extracts from wild-type cells after EDTA treatment or from cells containing Ntp1p with mutations in aspartic acid residues within the Ca2+-binding site revealed that Ntp1p eluted mainly in an inactive conformation instead of the dimeric or trimeric active form of the enzyme. These results suggest that activation of S. pombe Ntp1p under different conditions depends upon Ca2+ binding through the Ca2+-binding motif as a prerequisite for correct enzyme oligomerization to its active form. Given the high degree of conservation of the Ca2+ accommodation site, this might be a general mechanism regulating neutral trehalase activity in other yeasts and filamentous fungi.

Amino Acid Motifs↗
PubMed1984

Exo-(1----3)-beta-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans.

Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.

Amidohydrolases↗
PubMed1997

A neutral trehalase gene from Candida albicans: molecular cloning, characterization and disruption.

A neutral trehalase gene, NTC1, from the human pathogenic yeast Candida albicans was isolated and characterized. An ORF of 2724 bp was identified encoding a predicted protein of 907 amino acids and a molecular mass of 104 kDa. A single transcript of approximately 3.2 kb was detected by Northern blot analysis. Comparison of the deduced amino acid sequence of the C. albicans NTC1 gene product with that of the Saccharomyces cerevisiae NTH1 gene product revealed 57% identity. The NTC1 gene was localized on chromosome 1 or R. A null mutant (delta ntc1/delta ntc1) was constructed by sequential gene disruption. Extracts from mutants homozygous for neutral trehalase deletion had only marginal neutral trehalase activity. Extracts from heterozygous mutants showed intermediate activities between extracts from the wild-type strain and from the homozygous mutants. The null mutant showed no significant differences in pathogenicity as compared to the wild-type strain in a mouse model of systemic candidiasis. This result indicates that the neutral trehalase of C. albicans is not a potential target for antifungal drugs.

Amino Acid Sequence↗
PubMed1999

Purification of the trehalase GMTRE1 from soybean nodules and cloning of its cDNA. GMTRE1 is expressed at a low level in multiple tissues.

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside), a disaccharide widespread among microbes and lower invertebrates, is generally believed to be nonexistent in higher plants. However, the recent discovery of Arabidopsis genes whose products are involved in trehalose synthesis has renewed interest in the possibility of a function of trehalose in higher plants. We previously showed that trehalase, the enzyme that degrades trehalose, is present in nodules of soybean (Glycine max [L.] Merr.), and we characterized the enzyme as an apoplastic glycoprotein. Here we describe the purification of this trehalase to homogeneity and the cloning of a full-length cDNA encoding this enzyme, named GMTRE1 (G. max trehalase 1). The amino acid sequence derived from the open reading frame of GMTRE1 shows strong homology to known trehalases from bacteria, fungi, and animals. GMTRE1 is a single-copy gene and is expressed at a low but constant level in many tissues.

Amino Acid Sequence↗