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Acrylonitrile inhibited trehalase and phosphorylases in larvae and adults of Tribolium castaneum. In Trogoderma granarium larvae phosphorylases alone were inhibited. Acetylcholinesterase was not affected.
The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing. The most severe phenotype corresponded to the Delta nth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2). Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.
A three step purification procedure for trehalase from Saccharomyces cerevisiae with a recovery of 76% of the original activity is presented. The enzyme was activated by a heat shock treatment prior to homogenization of the cells. A mutant strain deleted in SUC genes was used to avoid contamination by invertase. The lyophylized enzyme was stable for, at least, 5 months and could be used to determine trehalose in the range 25 to 500 nmol. The preparation was free of inspecific phosphatases allowing for trehalose determinations in yeast cell free extracts and in insect hemolymph.
ALPHA,ALPHA-Trehalase (EC 3.2.1.28, alpha,alpha-trehalose glucohydrolase) was solubilized from the microvillous membrane of the intestinal mucosa of rats with Triton X-100 and butanol. It was purified 6350-fold by gel filtration on Sephadex G-150 and chromatography on DE-52 and hydroxyapatite. The purified enzyme, with a specific activity of about 127 units per mg of protein, showed almost a single band of protein and activity on polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 96 000 on Sephadex G-150 and 90 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its pH optimum was 5.5-5.7 and its Km value for trehalose was 5.4 mM. Its activity was inhibited 30 and 100% by 1 mM p-chloromercuribenzoate and 0.1 mM HgCl2, respectively and 30% by 1 mM MgCl2. Moreover, its activity was inhibited completely by 10 mM tris(hydroxymethyl)aminomethane and about 60% by 10 mM sucrose and cellobiose. The enzyme showed a high specificity for trehalose.
Carbodiimide modification of the Rhynchosciara americana midgut trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) at different pH values revealed the existence of two essential groups (pKa 5.28 and pKa 7.74) for the trehalos activity. Those groups must be carboxyl groups since the alternative possibilities (sulfhydryl and phenol groups) have been discarded by selective modification and attempts to reactivate the modified enzyme with hydroxylamine. Furthermore, the increase of the pKa values of carbodiimide-reactive groups in the presence of dioxane supports further evidence that they are carboxyls. The results suggest the pKa 5.28 carboxyl is in the active site, while the pKa 7.74 carboxyl is in its neighborhood buried in the enzyme molecule. The possible role for the carbodiimide-reactive carboxyl groups in catalysis is discussed.
Reaction of (+/-)-(3/4,5,6)-4-bromo-5,6-epoxy-3-hydroxycyclohexene with 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-glucopyranose, followed by treatment of the resulting isolated diastereoisomeric 4-bromo-3,5-dihydroxycyclohexene 1-thioglycoside derivatives with base under phase-transfer conditions, gave (R)- and (S)-(3,4,6/5)-3,4-epoxy-6-S-(1-thio-alpha-D-glucopyranosyl)-5- hydroxycyclohexene. None of them was substrate or inhibitor for cockchafer trehalase.
The disaccharide trehalose plays a critical role in yeast cell survival during conditions of environmental stress. The vacuole of the yeast Saccharomyces cerevisiae contains an enzyme, acid trehalase (ATH), that is capable of degrading trehalose. Recently, a gene required for ATH activity, ATH1, was cloned and sequenced [Destruelle et al., (1995) Yeast 11, 1015-1025]. The relationship between ATH1 and ATH, however, was not determined. We have purified ATH and shown that it is the ATH1 gene product; peptide sequences from the purified protein correspond to the deduced amino acid sequence of Ath1p. In addition, antiserum to Ath1p specifically recognizes purified ATH.
An extracellular trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) was purified from conidia of Humicola grisea var. thermoidea. The purified enzyme is a glycoprotein and migrates as a single polypeptide band during polyacrylamide gel electrophoresis under non-denaturing conditions. The apparent molecular weight of the enzyme was estimated as 580,000 by gel filtration chromatography. The enzyme is separable into three polypeptide bands of 105,000, 98,000 and 84,000 daltons on SDS-PAGE. It is specific for trehalose and its activity is not inhibited by other disaccharides. It has a Km of 2.3 mM, an optimum pH of 5.6 in sodium acetate buffer and a temperature optimum of 60 degrees C. The enzyme is activated by Ca2+, Co2+ and Mn2+ and inhibited by inorganic phosphate, AMP, ADP or ATP. The inhibitory effect of phosphate, AMP and ADP, but not that of ATP, was abolished in the presence of Ca2+.
We have identified a sequencing error in the neutral trehalase-encoding gene NTH1 [Kopp et al., J. Biol. Chem. 268 (1993) 4766-4774]. This error extends the deduced amino acid (aa) sequence at the N terminus by 58 aa. The biological implications of this include the presence of an additional phosphorylation site, which is believed to regulate trehalose hydrolysis.
The midgut trehalase (THA) from fifth instar Lymantria dispar (gypsy moth) larvae was purified to homogeneity by two separate methods: gel filtration followed by Rotofor preparative IEF, and affinity chromatography on trehalose coupled to Sepharose 6B followed by preparative polyacrylamide gel electrophoresis. Midgut THA from the last stadium L. dispar larvae existed mainly in soluble form and displayed a single band of activity in nondenaturing polyacrylamide gels when stained by a THA-specific staining procedure. Analytical IEF of purified midgut THA revealed a single protein band with an apparent pI of 4.6. SDS-PAGE and gel permeation studies indicated that the smallest active form of THA in the late fifth instar larval midgut was a monomeric protein with an approximate size of 60 kDa. A specific activity of 67 units/mg of protein at 30 degrees C and at pH 6.4 was determined for the enzyme purified by affinity chromatography and preparative gel electrophoresis. The midgut enzyme exhibited a very high substrate specificity with a Km of 0.4 mM for trehalose. The enzyme was maximally active at pH 5.4-6.0 and was thermally stable at temperatures up to 65 degrees C. The midgut THA was insensitive to inhibition by a high concentration of Tris, sucrose, p-nitrophenyl-beta-D-glucoside or phloridzin. Divalent cations metal ions, hypertrehalosaemic hormone and octopamine had no significant effect on the activity of the purified enzyme in vitro. The purified enzyme was inactivated by modification with DEP and was competitively inhibited by castanospermine with an apparent Ki of 0.8 x 10(-6)M at pH 6.4.
Eight analogues of trehazolin were synthesized and tested for trehalase inhibitors. Deoxygenation of the cyclopentanepolyol moiety all decreased the inhibitory activity. Epimerization at the branching point of the cyclopentane ring did not so affect the potency. The cyclic isourea part was shown to be replaced with guanidine structure with a considerable decrease of activity. The 6'-fluoro-6'-deoxy derivative was still a strong inhibitor. It seems that trehazolin strictly mimies the substrate alpha, alpha-trehalose and any structural change and/or removal of the hydroxyl functions appreciably influence its potency. The present results led to finding 5-aminocyclopentane-1,2,3,4-tetraols to be new lead compounds for glycohydrolase inhibitors.
An acid trehalase from Rhizopus microsporus var. rhizopodiformis was purified to apparent homogeneity. The molecular weight by SDS-PAGE (60 kDa) or Sephacryl S-200 filtration (105 kDa) suggested a homodimer. The carbohydrate content was 72%. Endoglycosidase H digestion resulted in one sharp band of 51.5 kDa in SDS-PAGE. pH and temperature optima were 4.5 and 45 degrees C, respectively. The isoelectric point was 6.69 and activation energy was 1.14 kcal mol(-1). The enzyme was stable for 1 h at 50 degrees C and decayed at 60 degrees C (t50 of 1.3 min.). Apparent KM for trealose was 0.2mM. Immunolocalisation studies showed the enzyme tightly packed at the surface of the cells.
Twelve N-arylglycosylamines were prepared in a one-pot reaction by treatment of D-glucose, D-galactose, D-mannose or D-xylose with fluorinated anilines in the presence of small amount of hydrochloric acid. The inhibitory activity against porcine trehalase and the fungicidal activity of the title compounds toward Rhizoctonia solani have been tested.
Acid trehalase (AT) has always been reported to be copurified with invertase (I) and a 40 kDa additional protein. Glucose grown stationary phase cells of Saccharomyces cerevisiae contained least I activity. So, it was attempted to purify AT from these cells (I:AT = 10.83). Studies on specific activity, percent recovery and I:AT ratio of different pools, collected during purification of AT, indicated that samples containing ratio I:AT < 2.2 were unstable. Purification methodology favouring association (DEAE-Sephadex chromatography) resulted in gaining total activity while methodology favouring dissociation (HPGPLC) resulted in tremendous loss in recovery. Active pool (Pool 1X) appeared to be electrophoretically homogeneous but dissociated into 175, 90, 68, 61, 57 (minor bands) and 37-41 (major band) molar mass (kDa) bands on SDS-PAGE. Inactive pools (Pools 1Y, 3X, 3Y) did not contain the 37-41 kDa major band. So, association of both I and a 37-41 kDa protein with AT appeared to be essential. Two bands of isoelectric pH (pI) 4.6 and 4.7 were present in pool 1X enzyme preparation. All SDS-PAGE-resolved bands of pool 1X, in an average, contained high aspartate/asparagine and low cysteine residues. AT activity appeared to be highly sensitive to the change in pH and also to agents affecting ionisation of protein, e.g., betaine, NaCl, acetate, etc. Association of AT components in presence of NaCl was demonstrated spectrophotometrically. Specific activity of AT decreased with dilution. Substrate mediated allosterism for this enzyme preparation suggested that AT existed as an equilibrium mixture of protomer-oligomer. It was suggested that reversible association-dissociation was a mechanism for the regulation of AT activity.
1. The effects of Ca(2+) on the activities and regulatory properties of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from vertebrate red and white muscle and insect fibrillar and non-fibrillar muscle have been investigated. These muscles were selected because of the possible difference in the role of glycolysis in energy production in the vertebrate muscles, and the possible difference in the role of Ca(2+) in the control of contraction in the two types of insect muscle. An increase in Ca(2+) concentration from 0.001mum to 10mum did not modify the activities nor did it modify the regulatory properties of these enzymes from these various muscles. 2. Concentrations of Ca(2+) above 0.1mm inhibited the activities of hexokinase and phosphofructokinase from the different muscles. It has been suggested that this inhibition may provide the basis for a theory of regulation of glycolysis (Margreth et al., 1967). If phosphofructokinase is located within the sarcoplasmic reticulum, its activity will be inhibited when the muscle is at rest, but the release of Ca(2+) from the reticulum during contraction will lead to a stimulation of its activity and hence an increase in glycolytic flux. The distribution of hexokinase and phosphofructokinase in the various cell fractions of these muscles was very variable. In particular, both enzymes were present almost exclusively in the 100000g supernatant fraction in the extracts of insect flight muscles. Thus there is no correlation between the properties of the enzymes and their distribution in muscle. 3. It is concluded that Ca(2+) does not control the activities of the important regulatory enzymes of glycolysis in muscle. It is suggested that in some muscles the sensitivity of the control mechanism at the level of phosphofructokinase to changes in the concentration of AMP may be increased by a process known as ;substrate-cycling'.
Trehalase activities at pH 5 (not inhibited by EDTA) and pH 7 (inhibited by EDTA) were present in the soluble fraction of disintegrated commercial baker's yeast. The pH 5 activity binds strongly to concanavalin A, is only partially salted out by saturated (NH4)2SO4, has an apparent Mr of 215000 (by gel filtration) and is an acidic protein. It has a Km of 1.4 mM, a broad pH optimum (at 40 mM-trehalose) between pH 4 and 5, and is activated by about 30% by 20-300 mM neutral salts such as KCl, NaNO3 and MnCl2. The enzyme is strongly inhibited by acetic acid/acetate buffers, with a Ki of about 15 mM-acetic acid. The pH 7 activity does not bind to concanavalin A, is salted out at 20-32% (w/v) (NH4)2SO4 and has an Mr of 170000 (by gel filtration). It is absolutely dependent on Ca2+ or Mn2+ ions (Mg2+ is ineffective) and strongly inhibited by neutral salts in the 20-100 mM range. It can be activated by treatment with MgATP in the presence of cyclic AMP. Activation decreases, but does not abolish, the Ca2+ requirement, and does not change the Km for trehalose (5.7 mM) or shift the sharp pH optimum at pH 6.7 (at 40 mM-trehalose).
Phosphoglucomutase (PGM) and trehalase (Tre) isoenzymes of five species of Simulium blackflies (Diptera: Simuliidae), vectors of onchocerciasis in Venezuela, were investigated by means of a portable electrophoresis field kit. Tre differed between S. incrustatum and S. oyapockense s.l. Electrophoretic variation of Tre in other members of the S. amazonicum and S. incrustatum groups merit further investigation. PGM appears to be more useful for separating populations within species complexes. Multiple populations and/or seasonal changes in population structure of S. guianense s.l., S. exiguum s.l. and S. metallicum s.l. were inferred from elecrophoretic variation of PGM.
One isoform of trehalase, TreF, is present in the cytoplasm and a second, TreA, in the periplasm. To study the questions of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular compartment, we fused the signal sequence of periplasmic TreA to cytoplasmic TreF. Even though this TreF construct was exported efficiently to the periplasm, it was not active. It was insoluble and degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre(+) revertants of periplasmic TreF. To further characterize periplasmic TreF, we used a genetic selection to isolate functional TreA-TreF hybrids, which were analyzed with respect to solubility and function. These data suggested that a domain located between residues 255 and 350 of TreF is sufficient to cause folding problems in the periplasm. In contrast to TreF, periplasmic TreA could fold into the active conformation in its nonnative cellular compartment, the cytoplasm, after removal of its signal sequence.