Trehalase from
            <i>Dictyostelium discoideum</i>
            : Purification and Properties

Ceccarini, Costante

doi:10.1126/science.151.3709.454

Cited by 42 publications

(3 citation statements)

References 9 publications

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“…However, in addition to trehalose 6-phosphate synthetase, myxamoebae also contain the enzyme trehalase which will degrade trehalose to glucose (Ceccarini, 1966). Thus increased trehalose accumulation could be the result of decreased cellular trehalase activity.…”

Section: Control Of End-product Saccharide Synthesismentioning

confidence: 99%

The control of saccharide synthesis during development of myxamoebae of Dictyostelium discoideum containing differing amounts of glycogen

Hames

Ashworth

1974

Biochemical Journal

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1. Myxamoebae initially containing 5.59mg of glycogen/10(8) cells accumulate approx. 25% more cell-wall polysaccharide, 100% more mucopolysaccharide, 200% more glucose and 300% more trehalose during their development than do myxamoebae initially containing less than 0.3mg of glycogen/10(8) cells. 2. These observations restrict the number of possible control mechanisms operating to regulate carbohydrate metabolism during development. 3. Cells accumulating a large amount of trehalose (approx. 400mug/10(8) cells) have the same amount and pattern of changes in specific activity of trehalase and trehalose 6-phosphate synthase as do cells accumulating a smaller amount of trehalose (approx. 100mug/10(8) cells). 4. These two populations of cells do, however, differ markedly in the amount of UDP-glucose and glucose 6-phosphate that they contain. 5. It is concluded that this change in the intracellular pools of the metabolic precursors of trehalose accounts for the increased amount of trehalose synthesized by cells derived from myxamoebae containing an increased glycogen content.

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Section: Control Of End-product Saccharide Synthesismentioning

confidence: 99%

The control of saccharide synthesis during development of myxamoebae of Dictyostelium discoideum containing differing amounts of glycogen

Hames

Ashworth

1974

Biochemical Journal

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“…The enzyme solutions tested produced a constant activity with time as well as a direct relationship between protein concentration and production of reducirng sugar. The Michaelis constant of the purified enzyme for trehalose, calculated by the method of Lineweaver and Burk (10), was 3.2 x 10-3 M, which is about the order of magnitude reported for vertebrate and mammalian trehalases (2, 17)' and trehalases from other sources (1,4). The pH optimum of the enzyme was 4.0, appreciably lower than that of most trehalases reported to date (1,4,5,7,8,17).…”

mentioning

confidence: 75%

Isolation and Identification of Trehalase from Pullularia pullulans

1971

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Trehalase has been isolated from Pullularia pullulans. The enzyme, which is specific for trehalose, was purified approximately 800-fold. The optimal pH was found to be 4.0 and the Michaelis dissociation constant, Km. was determined to be 3.2 x 10-3 M. Trehalose has been found in microorganisms, vertebrates, invertebrates, and phanerogams (15). In fungi, trehalose functions as a reserve carbohydrate sustaining respiration and accelerating germination (6,7,14). Trehalose has recently been isolated from the yeastlike fungus Pullularia pullulans in which its accumulation parallels the increase of pigment formation in the cells (12,13). P. pullulans is exceedingly common and found on fruits, vegetables, meat products, and cheese and is involved in the deterioration of paint and discoloration of lumber. Thus, it seemed of interest to undertake the isolation and characterization of trehalase from these cells. P. pullulans (strain NRRL YB-4515) was grown in a medium consisting of (Difco) 0.3% malt and yeast extracts, 1.5% glucose, 0.0135% K2HPO4, 0.01 % MgSO4-7H20, 0.01% KH2PO4, and 0.02% NH,Cl. After 5 days of shaking on a rotary shaker (220 rev/min and 2.5-inch thrust) at 28 C, the cells were harvested by centrifugation and washed first with 1% NaCI solution and then with distilled water until free from chloride. The cells were broken by adding to each gram of cells 3 g of 0.1-mm glass beads and 3 ml of 0.1 M sodium acetate buffer (pH 5.5) in a Sorvall Omni-mix. The rupturing was performed at 5 C at a rheostat setting of 100 for I hr, at which point microscopic examination indicated 80 to 90% breakage. The supernatant fluids were then centrifuged at 1,370 x g for 10 min, and the resulting residue was washed several times with 0.1 M acetate buffer (pH 5.5), hereafter referred to as "standard buffer." The washings were combined with the supernatant fluids, and the combined mixture was centrifuged at 6,590 x g for 10 min followed by centrifugation of the supernatant at 105,400 x g for 60 min. Enzyme activity was measured by employing samples of the fractions mixed with I ml of sugar solution containing 10 ,umoles of trehalose dissolved in standard buffer. After 15, 30, 45, and 60 min of incubation at 37 C, the amount of reducing sugar was determined by the Folin-Wu method (3). A unit of enzyme activity is defined as that amount of enzyme which produced 1.0 ,umole of glucose per min per mg of protein at 37 C. Protein content of the enzyme solutions was determined by the procedure of Lowry et al. (1 1).Ammonium sulfate was added to the supernatant liquid containing the enzyme with stirring at 5 C to a concentration of 40% saturation. After removal of the precipitate by centrifugation, the addition of ammonium sulfate was continued until a 70% saturation was obtained. After separation of the precipitate by centrifugation (20,000 x g), the supernatant was dialyzed against distilled water until free from ammonium sulfate. A small portion of this solution was dialyzed against standard buffer, whereas the remaining portion wa...

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“…It is interesting to note that an acidic condition is usually optimal 100 MYCEULUM /cP STIPE . 5 X0\.X NI, 25 3. for trehalase in fungi (12,18,27,32), although a hybrid yeast exhibits maximal trehalase activity at pH 6.9 (1). Multiple forms of trehalase may also have the same pH optimum, as is the case in Neurospora crassa (18).…”

Section: Discussionmentioning

confidence: 99%

Carbohydrate Metabolism During Morphogenesis of Coprinus lagopus ( sensu Buller)

1969

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The occurrence and properties of enzymes of carbohydrate metabolism were studied during dikaryotic fruiting of the mushroom Coprinus lagopus. Enzymes of hexose monophosphate catabolism, sugar alcohol (polyol) dehydrogenases (DH), and trehalase occurred throughout development. The ratio of xylitol DH to sorbitol DH was greater than unity in both monokaryotic mycelium and dikaryotic fruit body caps, whereas this ratio decreased in the stipe (stalk) tissue. Xylitol DH and sorbitol DH were both dependent upon nicotinamide adenine dinucleotide (NAD) and showed maximal activity at pH 9. Two separate enzymes were suspected on the basis of preferential utilization of the NAD analogue, thionicotinamide-NAD, by xylitol DH, and this feature was consistent throughout development. An appraisal of the carbohydrate pool revealed trehalose and glucose, with the former predominant in the stipe and the latter in excess in the cap of dikaryotic fruit bodies. Trehalase activity in dialyzed enzyme extracts showed pH optima at acid and alkaline pH levels in monokaryotic mycelium, dikaryotic stipes, and cap tissues.Developmental studies of the fruiting basidiomycetous fungus Coprinus have provided basic information relative to general problems of microbial morphogenesis (9). Vegetative monokaryotic hyphae ordinarily produce asexual propagules called oidia. These uninucleate cells may either germinate to produce monokaryotic hyphae or serve as fertilizing elements for compatible vegetative hyphae (6-8). Mating occurs by hyphal anastomosis and nuclear exchange between monokaryons bearing dissimilar A and B incompatibility factors to produce a dikaryon containing clamp connections. Profound morphological changes in filamentous dikaryotic hyphae produce a gilled fruitbody consisting of a prominent stipe (stalk) and cap. Diploidization and meiosis in the basidium are followed by basidiospore production. The mature fruit body cap then undergoes autolysis and basidiospore dissemination.Much past research dealing with sporogenesis in Coprinus has been descriptive. The origin of lamellae was outlined in fruit bodies of Coprinus micaceus (19), and growth kinetics were defined during fruiting of C. lagopus (4). Later work by Madelin (23) showed that reserves were drawn from vegetative mycelium, presumably to sustain fruit body development in C. lagopus, and that these stores may originate in swollen hyphae containing glycogen and protein (25). The effects of light and temperature (5,16,24) and the role of the nutritional milieu have also been clarified (2, 10, 30). Further insight was gained from electron microscopy studies of the hymenium of sporulating fruit bodies of C. lagopus; the occurrence and morphological details of synaptinemal complexes (21, 22) were correlated with genetic evidence for meiosis (cf. 15).In contrast to this sort of information, little is known about the physiological changes accompanying morphogenesis of C. lagopus. Accordingly, biochemical studies were initiated concerning the intermediary metabolism of carbohydrates d...

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Trehalase from Dictyostelium discoideum : Purification and Properties

Cited by 42 publications

References 9 publications

The control of saccharide synthesis during development of myxamoebae of Dictyostelium discoideum containing differing amounts of glycogen

The control of saccharide synthesis during development of myxamoebae of Dictyostelium discoideum containing differing amounts of glycogen

Isolation and Identification of Trehalase from Pullularia pullulans

Carbohydrate Metabolism During Morphogenesis of Coprinus lagopus ( sensu Buller)

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