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Solomon Nwaka - One of the best experts on this subject based on the ideXlab platform.
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evidence for contribution of neutral Trehalase in barotolerance of saccharomyces cerevisiae
Applied and Environmental Microbiology, 2000Co-Authors: Hitoshi Iwahashi, Solomon Nwaka, Kaoru ObuchiAbstract:In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral Trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral Trehalase in barotolerance. To estimate the contribution of neutral Trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral Trehalase I and/or neutral Trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral Trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral Trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral Trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral Trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral Trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral Trehalase contributes to barotolerance, especially during recovery.
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opposite roles of Trehalase activity in heat shock recovery and heat shock survival in saccharomyces cerevisiae
Biochemical Journal, 1999Co-Authors: Stefaan Wera, Solomon Nwaka, Ellen De Schrijver, Ilse Geyskens, Johan M TheveleinAbstract: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.
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Neutral Trehalases Contribute to Barotolerance in Saccharomyces cerevisiae
Advances in High Pressure Bioscience and Biotechnology, 1999Co-Authors: Hitoshi Iwahashi, Solomon Nwaka, Kaoru Obuchi, Yasuhiko KomatsuAbstract:To estimate the contribution of neutral Trehalases to barotolerance, we measured barotolerance of neutral Trehalase I and/or neutral Trehalase II deficient strains. Under 180 MPa for 2 h, the neutral Trehalase I deficient strain showed lower barotolerance than the wild type strain. The neutral Trehalase II deficient strain did not show lower barotolerance than the wild type strain. However, the neutral Trehalase I and neutral Trehalase II double mutant showed much lower barotolerance than the neutral Trehalase II deficient strain. These results suggest that neutral Trehalase I directly contributes to barotolerance and that neutral Trehalase II may indirectly contribute to barotolerance. To confirm the direct contribution of neutral Trehalase I, we transformed the neutral Trehalase I deficient strain with the plasmid which contained neutral Trehalase I gene. This transformant showed higher barotolerance than the control strain which carried the plasmid without neutral Trehalase I gene. Our data suggest that neutral Trehalase I has a direct role in barotolerance.
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neutral Trehalase nth1p of saccharomyces cerevisiae encoded by the nth1 gene is a multiple stress responsive protein
FEBS Letters, 1997Co-Authors: Harald Zahringer, Mark Us Burgert, Helmut Holzer, Solomon NwakaAbstract:We have shown previously that expression of the NTH1 gene is increased at heat stress (40°C) both at the mRNA and enzymatic activity levels. This increased expression was correlated to the requirement of the NTH1 gene for recovery after heat shock at 50°C and the presence of stress responsive elements STRE (CCCCT) 3 times in its promoter region [S. Nwaka et al., FEBS Lett. 360 (1995) 286–290; S. Nwaka et al., J. Biol. Chem. 270 (1995) 10193–10198]. We show here that expression of the NTH1 gene and its product, neutral Trehalase (Nth1p), are also induced by other stressors such as H 2 O 2 , CuSO 4 , NaAsO 2 , and cycloheximide (CHX). Heat-induced expression of the NTH1 gene is shown to be accompanied by accumulation of trehalose. In contrast, the chemical stressors which also induce the expression of NTH1 did not lead to accumulation of trehalose under similar conditions. Our data suggest that: (1) heat- and chemical stress-induced expression of neutral Trehalase is largely due to de novo protein synthesis, and (2) different mechanisms may control the heat- and chemical stress-induced expression of NTH1 at the transcriptional level. Participation of neutral Trehalase (Nth1p) in multiple stress response dependent and independent on trehalose is discussed.
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Molecular Biology of Trehalose and the Trehalases in the Yeast Saccharomyces cerevisiae
Progress in Nucleic Acid Research and Molecular Biology, 1997Co-Authors: Solomon Nwaka, Helmut HolzerAbstract:The present state of knowledge of the role of trehalose and trehalose hydrolysis catalyzed by Trehalase (EC 3.2.1.28) in the yeast Saccharomyces cerevisiae is reviewed. Trehalose is believed to function as a storage carbohydrate because its concentration is high during nutrient limitations and in resting cells. It is also believed to function as a stress metabolite because its concentration increases during certain adverse environmental conditions, such as heat and toxic chemicals. The exact way trehalose may perform the stress function is not understood, and conditions exist under which trehalose accumulation and tolerance to certain stress situations cannot be correlated. Three Trehalases have been described in S. cerevisiae: 1) the cytosolic neutral Trehalase encoded by the NTH1 gene, and regulated by cAMP-dependent phosphorylation process, nutrients, and temperature; 2) the vacuolar acid Trehalase encoded by the ATH1 gene, and regulated by nutrients; and 3) a putative Trehalase Nth1p encoded by the NTH2 gene (homolog of the NTH1 gene) and regulated by nutrients and temperature. The neutral Trehalase is responsible for intracellular hydrolysis of trehalose, in contrast to the acid Trehalase, which is responsible for utilization of extracellular trehalose. The role of the putative Trehalase Nth2p in trehalose metabolism is not known. The NTH1 and NTH2 genes are required for recovery of cells after heat shock at 50 °C, consistent with their heat inducibility and sequence similarity. Other stressors, such as toxic chemicals, also induce the expression of these genes. We therefore propose that the NTH1 and NTH2 genes have stress-related (unction and the gene products may be called stress proteins. Whether the stress function of the Trehalase genes is linked to trehalose is not clear, and possible mechanisms of stress protective function of the Trehalases are discussed. © 1998 Academic Press
Hiroyuki Takano - One of the best experts on this subject based on the ideXlab platform.
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stress tolerance in doughs of saccharomyces cerevisiae Trehalase mutants derived from commercial baker s yeast
Applied and Environmental Microbiology, 1999Co-Authors: Jun Shima, Ryouichi Nakajima, Katsumi Mori, Hajime Watanabe, Akihiro Hino, Yasuo Suzuki, Chie Yamadaiyo, Hiroyuki TakanoAbstract:Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral Trehalase mutants (Δnth1), acid Trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the Trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the Trehalase mutants may make these strains useful in frozen dough.
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Stress tolerance in doughs of Saccharomyces cerevisiae Trehalase mutants derived from commercal baker's yeast
Applied and Environmental Microbiology, 1999Co-Authors: Jun Shima, Ryouichi Nakajima, Chie Yamada-Iyo, Katsumi Mori, Hajime Watanabe, Akihiro Hino, Yasuo Suzuki, Hiroyuki TakanoAbstract:Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral Trehalase mutants (Deltanth1), acid Trehalase mutants (Deltaath1), and double mutants (Deltanth1 ath1) by using commercial baker's yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the Trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Deltanth1 and Deltaath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Deltanth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the Trehalase mutants may make these strains useful in frozen dough.
Andres Wiemken - One of the best experts on this subject based on the ideXlab platform.
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induction of Trehalase in arabidopsis plants infected with the trehalose producing pathogen plasmodiophora brassicae
Molecular Plant-microbe Interactions, 2002Co-Authors: David Brodmann, Roger A Aeschbacher, Thomas Boller, Andres Wiemken, Astrid Schuller, Jutta Ludwigmuller, Astrid WinglerAbstract:Various microorganisms produce the disaccharide trehalose during their symbiotic and pathogenic interactions with plants. Trehalose has strong effects on plant metabolism and growth; therefore, we became interested to study its possible role in the interaction of Arabidopsis thaliana with Plasmodiophora brassicae, the causal agent of clubroot disease. We found that trehalose accumulated strongly in the infected organs (i.e., the roots and hypocotyls) and, to a lesser extent, in the leaves and stems of infected plants. This accumulation pattern of trehalose correlated with the expression of a putative trehalose-6-phosphate synthase (EC 2.4.1.15) gene from P. brassicae, PbTPS1. Clubroot formation also resulted in an induction of the Arabidopsis Trehalase gene, ATTRE1, and in a concomitant increase in Trehalase (EC 3.2.1.28) activity in the roots and hypocotyls, but not in the leaves and stems of infected plants. Thus, induction of ATTRE1 expression was probably responsible for the increased Trehalase activity. Trehalase activity increased before trehalose accumulated; therefore, it is unlikely that Trehalase was induced by its substrate. The induction of Trehalase may be part of the plant's defense response and may prevent excess accumulation of trehalose in the plant cells, where it could interfere with the regulation of carbon metabolism.
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trehalose and Trehalase in arabidopsis
Plant Physiology, 2001Co-Authors: Joachim Muller, Roger A Aeschbacher, Thomas Boller, Astrid Wingler, Andres WiemkenAbstract:Trehalase is ubiquitous in higher plants. So far, indications concerning its function are scarce, although it has been implicated in the detoxification of exogenous trehalose. A putative Trehalase gene, T19F6.15 , has been identified in the genome sequencing effort in Arabidopsis. Here we show that this gene encodes a functional Trehalase when its cDNA is expressed in yeast, and that it is expressed in various plant organs. Furthermore, we present results on the distribution and activity of Trehalase in Arabidopsis and we describe how inhibition of Trehalase by validamycin A affects the plants response to exogenous trehalose (α-d-glucopyranosyl-[1, 1]-α-d-glucopyranoside). Trehalase activity was highest in floral organs, particularly in the anthers (approximately 700 nkat g −1 protein) and maturing siliques (approximately 250 nkat g −1 protein) and much lower in leaves, stems, and roots (less than 50 nkat g −1 protein). Inhibition of Trehalase in vivo by validamycin A led to the accumulation of an endogenous substance that had all the properties of trehalose, and to a strong reduction in sucrose and starch contents in flowers, leaves, and stems. Thus, trehalose appears to be an endogenous substance in Arabidopsis, and trehalose and Trehalase may play a role in regulating the carbohydrate allocation in plants.
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purification of the Trehalase gmtre1 from soybean nodules and cloning of its cdna gmtre1 is expressed at a low level in multiple tissues
Plant Physiology, 1999Co-Authors: Roger A Aeschbacher, Joachim Muller, Thomas Boller, Andres WiemkenAbstract:Trehalose (α-d-glucopyranosyl-1,1-α-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 Trehalase1). 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.
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trehalose and Trehalase in plants recent developments
Plant Science, 1995Co-Authors: Joachim Muller, Thomas Boller, Andres WiemkenAbstract:Abstract Trehalose is a non-reducing disaccharide consisting of two α-glycosidically linked glucose units. It accumulates in many microorganisms and invertebrate animals when they are exposed to various forms of stress, and it may serve as a protectant of enzymes and membranes, particularly under conditions of heat and desiccation stress. Most vascular plants lack the capacity to produce trehalose, except for a small number of desiccation tolerant plants, such as some ferns and the angiosperm Myrothamnus flabellifolia . In contrast, a highly specific Trehalase activity has been described in many plants. The enzyme does not cleave other common α-glucosides, and it is highly sensitive to the inhibitor validamycin A. Trehalases have been found in various tissues; particularly high activities occur in pollen and legume root nodules. The possible functions of plant Trehalase are discussed, focussing on its significance in the interaction of plants with trehalose-accumulating microorganisms.
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effects of validamycin a a potent Trehalase inhibitor and phytohormones on trehalose metabolism in roots and root nodules of soybean and cowpea
Planta, 1995Co-Authors: Joachim Muller, Thomas Boller, Andres WiemkenAbstract:Trehalose, a common microbial disaccharide, has been reported to be toxic to plants, and plant Trehalase has therefore been hypothesized to function as a detoxifying enzyme. To test this, aseptically grown soybean (Glycine max L. Merr.) plantlets were supplied with trehalose. The plants accumulated trehalose only when validamycin A, a potent Trehalase inhibitor, was added as well. Under these conditions, they accumulated trehalose to up to 8% of the dry weight in their primary leaves without any detectable impairment of growth or health. We have previously shown that in soybean nodules, trehalose is generated by the symbiotic bacteria, and Trehalase is strongly induced. However, direct exposure of plants to trehalose did not affect their Trehalase activity, whereas a treatment with auxin strongly increased it, indicating that the enzyme level is regulated by hormones rather than by its substrate. Addition of validamycin A to nodules caused an increase in the amount of trehalose and a decrease in the sucrose and starch pools, but nitrogen fixation was not affected. Similar results were obtained with cowpea (Vigna unguiculata L.) plantlets and nodules. These results indicate that plant Trehalase is functional in metabolizing trehalose from exogenous and endogenous sources, even though the disaccharide has no obvious toxic effects.
Bin Tang - One of the best experts on this subject based on the ideXlab platform.
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knockdown of five Trehalase genes using rna interference regulates the gene expression of the chitin biosynthesis pathway in tribolium castaneum
BMC Biotechnology, 2016Co-Authors: Bin Tang, Lina Zhao, Qida Shen, Mengmeng Yang, Shigui WangAbstract:Background RNA interference is a very effective approach for studies on gene function and may be an efficient method for controlling pests. Trehalase is a key gene in the chitin biosynthesis pathway in insects. Five Trehalase genes have been cloned in Tribolium castaneum, though it is not known whether the detailed functions of these Trehalases can be targeted for pest control.
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two novel soluble Trehalase genes cloned from harmonia axyridis and regulation of the enzyme in a rapid changing temperature
Comparative Biochemistry and Physiology B, 2016Co-Authors: Qingye Xu, Shigui Wang, Su Wang, Fan Zhang, Bin TangAbstract:Abstract In previous studies, we have cloned two soluble Trehalase genes ( HaTreh1–1 and HaTreh1–2 ) from the harlequin ladybird Harmonia axyridis . Here, we obtained the other two novel genes ( HaTreh1–3 and HaTreh1–4 ) by transcriptome sequencing and rapid amplification of cDNA ends. Generally, anabolism enhancement and catabolism inhibition together contribute to accumulation of trehalose, and Trehalase is the key enzyme to start the catabolism of trehalose. To characterize the metabolism of trehalose in H. axyridis and how these Trehalase genes are regulated under cold stress conditions, a comparison of trehalose content and Trehalase levels in two different rapidly changing temperature environments was carried out to explore the regulation of these genes. We found that an accumulation of trehalose could be observed at 5 °C, 0 °C and − 5 °C and Trehalase was suppressed in these temperature points during a gradually cooling environment. Then, in a gradually warming environment, trehalose levels increased slightly from − 5 °C to 15 °C and then decreased at 25 °C; however, no significant negative association was observed between Trehalase and trehalose. Additionally, we found that glycogen could be converted into trehalose to help the individual resist the low temperature. Analysis of the expression of soluble Trehalase showed that HaTreh1–1 , HaTreh1–2 , HaTreh1–3 and HaTreh1–4 were involved in trehalose metabolism; but the gene HaTreh1–4 plays the most important role in the cooling process, and HaTreh1–2 and HaTreh1–4 play the most important role in the warming process. Finally, we found that 5 °C might be a temperature signal for H. axyridis ; prior to this temperature, individuals must make enough physical preparations to resist cold stress during the winter.
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different functions of the insect soluble and membrane bound Trehalase genes in chitin biosynthesis revealed by rna interference
PLOS ONE, 2010Co-Authors: Jie Chen, Bin Tang, Hongxin Chen, Xiaofeng Huang, Jing Chen, Daowei Zhang, Wenqing ZhangAbstract:Background Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, Trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) Trehalases. However, no functions of the two Trehalases in chitin biosynthesis in insects have yet been reported.
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characterization and expression patterns of a membrane bound Trehalase from spodoptera exigua
BMC Molecular Biology, 2008Co-Authors: Bin Tang, Xiaofei Chen, Honggang Tian, Jian Hu, Weihua Xu, Wenqing ZhangAbstract:Background The chitin biosynthesis pathway starts with trehalose in insects and the main functions of Trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble Trehalase (Tre-1) and membrane-bound Trehalase (Tre-2), very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2.
Helmut Holzer - One of the best experts on this subject based on the ideXlab platform.
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neutral Trehalase nth1p of saccharomyces cerevisiae encoded by the nth1 gene is a multiple stress responsive protein
FEBS Letters, 1997Co-Authors: Harald Zahringer, Mark Us Burgert, Helmut Holzer, Solomon NwakaAbstract:We have shown previously that expression of the NTH1 gene is increased at heat stress (40°C) both at the mRNA and enzymatic activity levels. This increased expression was correlated to the requirement of the NTH1 gene for recovery after heat shock at 50°C and the presence of stress responsive elements STRE (CCCCT) 3 times in its promoter region [S. Nwaka et al., FEBS Lett. 360 (1995) 286–290; S. Nwaka et al., J. Biol. Chem. 270 (1995) 10193–10198]. We show here that expression of the NTH1 gene and its product, neutral Trehalase (Nth1p), are also induced by other stressors such as H 2 O 2 , CuSO 4 , NaAsO 2 , and cycloheximide (CHX). Heat-induced expression of the NTH1 gene is shown to be accompanied by accumulation of trehalose. In contrast, the chemical stressors which also induce the expression of NTH1 did not lead to accumulation of trehalose under similar conditions. Our data suggest that: (1) heat- and chemical stress-induced expression of neutral Trehalase is largely due to de novo protein synthesis, and (2) different mechanisms may control the heat- and chemical stress-induced expression of NTH1 at the transcriptional level. Participation of neutral Trehalase (Nth1p) in multiple stress response dependent and independent on trehalose is discussed.
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Molecular Biology of Trehalose and the Trehalases in the Yeast Saccharomyces cerevisiae
Progress in Nucleic Acid Research and Molecular Biology, 1997Co-Authors: Solomon Nwaka, Helmut HolzerAbstract:The present state of knowledge of the role of trehalose and trehalose hydrolysis catalyzed by Trehalase (EC 3.2.1.28) in the yeast Saccharomyces cerevisiae is reviewed. Trehalose is believed to function as a storage carbohydrate because its concentration is high during nutrient limitations and in resting cells. It is also believed to function as a stress metabolite because its concentration increases during certain adverse environmental conditions, such as heat and toxic chemicals. The exact way trehalose may perform the stress function is not understood, and conditions exist under which trehalose accumulation and tolerance to certain stress situations cannot be correlated. Three Trehalases have been described in S. cerevisiae: 1) the cytosolic neutral Trehalase encoded by the NTH1 gene, and regulated by cAMP-dependent phosphorylation process, nutrients, and temperature; 2) the vacuolar acid Trehalase encoded by the ATH1 gene, and regulated by nutrients; and 3) a putative Trehalase Nth1p encoded by the NTH2 gene (homolog of the NTH1 gene) and regulated by nutrients and temperature. The neutral Trehalase is responsible for intracellular hydrolysis of trehalose, in contrast to the acid Trehalase, which is responsible for utilization of extracellular trehalose. The role of the putative Trehalase Nth2p in trehalose metabolism is not known. The NTH1 and NTH2 genes are required for recovery of cells after heat shock at 50 °C, consistent with their heat inducibility and sequence similarity. Other stressors, such as toxic chemicals, also induce the expression of these genes. We therefore propose that the NTH1 and NTH2 genes have stress-related (unction and the gene products may be called stress proteins. Whether the stress function of the Trehalase genes is linked to trehalose is not clear, and possible mechanisms of stress protective function of the Trehalases are discussed. © 1998 Academic Press
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phenotypic features of Trehalase mutants in saccharomyces cerevisiae
FEBS Letters, 1995Co-Authors: Solomon Nwaka, Bernd Mechler, Monika Destruelle, Helmut HolzerAbstract:Abstract In the yeast Saccharomyces cerevisiae , some studies have shown that trehalose and its hydrolysis may play an important physiological role during the life cycle of the cell. Recently, other studies demonstrated a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. We had reported lack of correlation between trehalose accumulation and increase in thermotolerance under certain conditions, suggesting that trehalose may not mediate thermotolerance [Nwaka, S., et al. (1994) FEBS Lett. 344, 225–228]. Using mutants of the Trehalase genes, NTH1 and YBR0106 , we have demonstrated the necessity of these genes in recovery of yeast cells after heat shock, suggesting a role of these genes in thermotolerance (Nwaka, S., Kopp, M., and Holzer, H., submitted for publication). In the present paper, we have analysed the expression of the Trehalase genes under heat stress conditions and present genetic evidence for the ‘poor-heat-shock-recovery’ phenotype associated with NTH1 and YBR0106 mutants. Furthermore, we show a growth defect of neutral and acid Trehalase-deficient mutants during transition from glucose to glycerol, which is probably related to the ‘poor-heat-shock-recovery’ phenomenon.
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assay of trehalose with acid Trehalase purified from saccharomyces cerevisiae
Yeast, 1993Co-Authors: Iris Kienle, Mark Us Burgert, Helmut HolzerAbstract: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°C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid Trehalase preparation used. As described previously (Mittenbuhler, K. and Holzer, H., 1988, J. Biol. Chem.263, 8537–8543; Mittenbuhler, 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.
