Trehalase and the enzymes of trehalose biosynthesis in Lilium longiflorum pollen

Gussin, Arnold E.S.; McCormack, Jeffrey H.

doi:10.1016/s0031-9422(00)85339-x

Cited by 30 publications

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References 18 publications

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“…Trehalose-6-phosphate synthetase (UDP-glucose:Dglucose-6-P 1-glucosyltransferase EC 2.4.1.15) assay was based on the method of Roth and Sussman (22). In the spectrophotometric assay the reaction mixture (0.8 ml) contained 100 mM Tris buffer (pH 7.5), 8 The radioassay for trehalose-6-P synthetase was based on the method of Lapp et al (13) The precipitate was washed with 0.4 ml of Tris buffer (pH 8.0), recentrifuged as above and the supernatant fluid was added to that from the first centrifugation. One unit of alkaline phosphatase was added and the tubes incubated for 2.5 h at 37°C.…”

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confidence: 99%

Enzymes of α,α-Trehalose Metabolism in Soybean Nodules

Salminen¹,

Streeter²

1986

Plant Physiol.

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Metabolism of trehalose, a,D-glucopyranosyl-a,D-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max IL.I Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and 0-glucose-l-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase.Trehalose, a-D-glucopyranosyl-a-D-glucopyranoside, is one of the major carbohydrates synthesized by cultured Bradyrhizobium japonicum, and every species of Rhizobium examined so far shows trehalose accumulation (26). This disaccharide has also been reported in yeast (1,5), in fungi (12, 22), as well as in bacteria (3), mainly in actinomycetes (15). In soybean plants trehalose appears to be restricted to the nodule tissue (23). Its presence elsewhere in higher plants has not been conclusively established (7). Previous work with soybean nodules suggests that trehalose is synthesized by the microsymbiont (20).The pathway for trehalose synthesis was first elucidated in Saccharomyces cerevisiae (5) (Fig. 1). The biosynthetic enzymes have also been reported in Lilium longiflorum pollen (8).There appears to be more than one way of catabolizing trehalose (Fig. 1). Phosphotrehalase has been reported in Bacillus popilliae (3) and trehalose phosphorylase in Euglena gracilis (17). Trehalase, the most widely occurring catabolic enzyme has also been found in higher plants (6,9,27 Nodule Fractionation. After 5 to 6 weeks the plants were harvested, and subsequently all operations were conducted at 0 to 2°C. Five g of nodules were ground in a mortar with a pestle in 10 ml (added in 2 ml aliquots) of grinding medium, 0.1 M sodium phosphate buffer (pH 7.5) containing 0.15 M mannitol, 2 mm DTE, and 1 mm EDTA. The homogenate was filtered through two Miracloth discs in a 50 ml syringe into a 15 ml centrifuge tube.The Miracloth filtrate was then centrifuged at 5OOg for 10 min. The resultant starch pellet was discarded and the supernatant fluid recentrifuged at 6,000g for 10 min to obtain the bacteroid pellet. The supernatant fluid was centrifuged at 48,000g for 15 min and the supernatant, cytosol fraction, was saved for gel filtration.The bacteroid pellet was resuspended in 8 ml of grindin...

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Enzymes of α,α-Trehalose Metabolism in Soybean Nodules

Salminen¹,

Streeter²

1986

Plant Physiol.

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“…Invertase I partially coeluted with a separate peak of trehalase activity on DEAE-Sephacel chromatography. Trehalase activity has been previously reponed in Lilium pollen (Gussin and McCormack, 1970).…”

Section: Discussionmentioning

confidence: 99%

Characterization and localization of three soluble invertase forms from Lilium longiflorum flower buds

Miller¹,

Ranwala²

1994

Physiol Plant

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Miller, W. B, and Ranwala. A. P. 1994. Characterization and localization of three soluble invertase forms from Lilium longiflorum flower buds. -Phvsiol. Plant. 92: 247-253.Three invertase forms (EC 3.2.1.26) were identified in soluble extracts from developing flower buds of Lilium longiflorum Tbunb. cv, Nellie White. The enzymes were separable on a diethylaminoethyl {DEAE)-Sephacel column and designated invertase I. II or III according to the order of elution from Sephacel. To determine tissue specificity of these floral invertases, anthers were separated from tepal. pistil and filament tissue, and analyzed for invertase acti\ ity. Invertase I was localized pnmarily in anthers, with invertases II and HI being preseni in much smaller amounts {less than 5% of the invertase I activity). Much higher ievels of invertases H and IJI were found in the nonanlher organs of the flower, where essentially no invena.se 1 was detectable. Further purification of each form (using gel filtration. Con-A-Sepharose affinity chromatography and hydrophobic interaction chromatography on pbenyl-agarose) resulted in 1.^5-. 189-and 2()2-fold purificairon of pooied fractions from DEAE-Sephacel. respectively, and established thai each invertase form is a glycoprotein. Each was an acid invertase. wiih pH optima between 4.0 and 5.0 and an apparent molecuiar mass of 77 500 Da (as detennined by Sephades gel filtration). The invertases had sucrose Kv alues of !.O. 6.4 and 6,6 mM. and temperature optima of 40. 50 and 45°C. re.speclively. A temperature stability study revealed that invertase III was the most themiostabie. followed by II and 1. Invertases II and 111 had lower affinity to raffinose and stachyose than invertase I, All three enzymes were completely inhibited by Hg-* or Ag* ions al 1.7 mM. .'\l this concentration. Cu-"' showed differential partial inhibition. .Although fructan was shoun to be present in both anther and nonanther tissues of Lilium llower bnds. these invertases showed no sucrose:sucrose fructosyitransferase (EC 2.4.1.991 activity, Ke\ words -Easter lily, flowering, fructan, fructofuranosidase, invertase. Lilium longiflorum. sucrose:sucrose fructosyllransferase.

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“…It was established that the value of Michaelis constant Km for trehalose was 0.25 mM at 37°C in sodium acetate buffer (pH 5.0), with the maximum velocity Vmax of 5667 pkat/mg protein. The catalytic properties of A. deliciosa wall-bound trehalase, such as the broad pH optimum, low Km and heat stability, resemble those of Lilium pollen (Gussin and Cormack, 1970) and Phaseolus nodule (García et al, 2005) enzymes, both of which are of plant origin.…”

Section: Substrate Specificity and Kinetic Parametersmentioning

confidence: 98%

Purification and Biochemical Characterization of Cell Wall-bound Trehalase from Pericarp Tissues of Actinidia deliciosa

Nakagawa

Sakurai

2008

J. Japan. Soc. Hort. Sci.

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A trehalase (EC 3.2.1.28) was purified from the cell walls of Actinidia deliciosa fruit. The purified trehalase had optimal pH of around 5, Km of 0.25 mM and Vmax of 5667 pkat/mg protein, and was relatively heat stable. The enzyme showed highly specific activity to trehalose and weak activity to maltose and maltotriose, but did not hydrolyze any other disaccharides. Trehalase activity was unaffected by Ca 2+ , Na, and Mg 2+ions and EDTA, but markedly inhibited by Hg 2+ and Fe 3+ ions, iodoacetic acid, tris(hydroxymethyl)aminomethane (Tris), p-chloromercuribenzoate (PCMB), glucose and glucosamine. This cell wall-bound enzyme seems to degrade apoplastic trehalose. Another possibility is that this trehalase has additional functions such as defence against insects.

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Trehalase and the enzymes of trehalose biosynthesis in Lilium longiflorum pollen

Cited by 30 publications

References 18 publications

Enzymes of α,α-Trehalose Metabolism in Soybean Nodules

Enzymes of α,α-Trehalose Metabolism in Soybean Nodules

Characterization and localization of three soluble invertase forms from Lilium longiflorum flower buds

Purification and Biochemical Characterization of Cell Wall-bound Trehalase from Pericarp Tissues of Actinidia deliciosa

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