Trehalose Synthesis during Differentiation in Dictyostelium discoideum

The synthesis of [%Z]trehalose has been studied in uiuo at various stages of differentiation in Dictyostelium discoideum. The rate of synthesis was negligible from aggregation until late in the culmination process. At that time, a lOO-fold burst of synthetic activity was observed, amounting to 0.04 pmole of trehalose per min per ml of packed cells. In the course of these studies radioactive maltose was also identified as a product of [14C]glucose metabolism.


ow114 SUMMARY
The synthesis of [%Z]trehalose has been studied in uiuo at various stages of differentiation in Dictyostelium discoideum. The rate of synthesis was negligible from aggregation until late in the culmination process.
At that time, a lOO-fold burst of synthetic activity was observed, amounting to 0.04 pmole of trehalose per min per ml of packed cells. In the course of these studies radioactive maltose was also identified as a product of [14C]glucose metabolism.
Trehalose is known to accumulate during differentiation in Dictyostelium discoideum (1,2). In the accompanying paper (3), kinetic models are presented which simulate the accumulation of this end product by various mechanisms.
These models predict that trehalose B-phosphate synthetase could not increase in activity in viva during the period in which in vitro analyses indicate that this synthetic enzyme does accumulate (i.e. prior to 800 min), and that the rate of trehalose synthesis in viva should be of the order of 0.03 pmole per min per ml of packed cells. In the present study, cells at various stages of differentiation were exposed to [%] Prior to use it was converted to the sodium salt.

Methods
Preparation of Cells D. discoideum strain NC-4 was grown according to the method of Liddel and Wright (4) except that the amoebae were respread onto cookie sheets containing 2% agar, 0.01 M phosphate buffer (pH 6.5) and 0.001 M EDTA.
The cells were allowed to recover at 23" for 2 hours and then were put at 15" overnight. Several hours before the desired stage of differentiation was reached, the cells were returned to 23", at which temperature the subsequent in vivo experiments were performed.

Labeling Conditions
The cells were harvested in Bonner's salt solution in 1O-3 M ME%NaOH, pH 6.5 (Bonner's MES), such that 1 ml of cell suspension contained 0.1 ml of packed cells. Three milliliters of the suspension were placed into 40-ml plastic round bottom centrifuge tubes and incubated in the presence of 0.02 mM [14C]glucose (1.8 x 10' dpm per pmole) for 10 to 40 min on a Fisher rotator (setting of 205). Separate series of tubes were used for the isolation of UDP-glucose, trehalose, and glucose-6-P.

Thin Layer Plates
Kiesetguhr G Plates for Trehalose Isolation-A slurry containing 20 g of Kieselguhr G plus 40 ml of 0.1 M phosphate, pH 6.0, was spread onto glass plates (20 x 20 cm) to a thickness of 250 rnp. Samples containing trehalose were streaked across the plates 2 cm from the edge of the plate and run ascending in l-butanol-acetone-O.1 M phosphate, pH 6.0 (4:5:1, v/v), to 14 1 The abbreviation used is: MES, 2-(N-morpholino)ethane sulfonic acid. 10,1971 D. Sargent and B. E. Wright 5341 cm above the origin. Each plate was run three times in the solvent, with drying between runs.

Issue of September
Unbound Cellulose Plates for Glucose-6-P Isolation-A slurry containing 12 g of h/Iacherey-Nagel MN300 unbound cellulose powder plus 72 ml of distilled water was spread onto glass plates (20 x 20 cm) to a thickness of 250 mp. Samples were run twice for 4 hours in 1-propanol-ammonia (sp. gr. 0.90)-water (6:3: 1, v/v) containing 2 g of EDTA per liter. Then the plates were run at right angles to the first dimension for 80 min in l-propylacetate-90y0 formic acid-water (11:5:3, v/v) (5).

UDP-glucose Isolation
At the desired time the cells were ruptured by the addition of an equal volume of cold 7% perchloric acid and the extraction and isolation were carried out as described previously (6).

Trehalose Isolation
The [%]glucose incorporation was stopped by diluting the cell suspension with ice-cold water and immediately centrifuging the cells at 1,900 x g, after which the supernatant solution was aspirated and the cells were extracted with 80% methanol for 30 to 60 min at room temperature (23"). It routinely took 6 min from dilution until the cells were extracted. Methanol was found to extract as much or more trehalose than treatment with 77, perchloric acid, chloroform-methanol (3:1, v/v) at 45", acetone at 45", or 80% ethanol.
Heating the cells to 100" in the presence of 80% methanol did not improve the extraction, The cell debris was centrifuged (14,500 x g for 10 min) and washed once with 80% methanol, and the supernatant solution plus wash was evaporated to dryness. The dried extract was dissolved in 1 ml of distilled water and centrifuged to remove some of the lipid, and the supernatant solution was streaked across the origin on a Kieselguhr G thin layer plate.
The lipid precipitste was washed once with 0.2 ml of distilled water and the wash was streaked over the origin.
The plates were run three times in the solvent system described, and then put with x-ray film for 7 to 10 days. Following the development of the films the trehalose bands were scraped and either counted directly in 10 ml of a dioxane gel (7) in a Beckman LS200 scintillation counter (85% efficiency with ['"Cl) or were eluted with 2 ml of distilled water for 30 min at room temperature, the Kieselguhr was washed, and the wash was added to the eluate. The samples were then desalted and enzyme-treated or the octaacetates were prepared.
A control experiment was carried out to determine whether Kieselguhr G acts as a quenching agent. It was found that 96 to 99% of known counts were recovered in the presence of an amount of Kieselguhr G comparable to that of a trehalose band.
Glucose-6-P Isolation Cells were either ruptured by the addition of an equal volume of cold 77, perchloric acid and worked up the same as the UDPglucose samples to the charcoal step, or they were extracted with 80% methanol and rinsed, the extract was evaporated to dryness, and the residue was dissolved in 1 ml of distilled water. The aqueous solution resulting from either method was extracted with chloroform three times and then chromatographed on Whatman No. 1 filter paper and run descending in l-butanol-ethanolacetone-water (5:4:3:2, v/v) for 40 hours, dried, and rerun in n-propyl acetate-go% formic acid-water (11:5:3, v/v) for 5 hours. The glucose-6-P area was cut out, eluted with distilled water, concentrated; and run two dimensions on unbound cellulose plates.
(See "Thin Layer Plates"). The plates were then put with x-ray film for 10 to 12 days, and the radioactive glucose-6-P area was scraped off and eluted.
The specific radioactivity was obtained by comparing the radioactivity of the spot with the micromoles of glucose-6-P present as determined by assay with glucose-6-P dehydrogenase. Phosphogluconic dehydrogenase was found not to be present in the glucose-6-P dehydrogenase preparation. Contamination with fructose 6-phosphate, which runs close to glucose-6-P in these solvents, was excluded since addition of phosphoglucose isomerase to the completed reaction mixture gave no further increase in glucose-6-P conversion.
Glucose l-phosphate is present at one-sixth the concentration of glucose-6-P and would represent a minor contamination (with a comparable specific radioactivity).

Preparation of Extracts for Trehalose-6-P Determination
Cells at preculmination were harvested in cold-distilled water and six 5-ml aliquots (packed cell volume of 0.59 ml/5 ml) were taken. The cells were either frozen and thawed before further treatment or were immediately treated as follows. Three aliquots were heated 10 min at loo", after which 0.25 mg of trehalose-6-P or 0.18 mg of glucose-6-P was added to one tube to serve as an internal control.
The precipitate was centrifuged and washed with distilled water, and the supernatant solution and wash were combined and evaporated to dryness.
The remaining three aliquots were extracted for 30 min at 23' in the presence of 2 volumes of 95% ethanol (8). Again, trehalose-6-P or glucose-6-P was added to one tube as an internal control.
Following extraction the cell debris was centrifuged, the precipitate was washed with 70% ethanol, and the supernatant solutions were evaporated to dryness.

Alkaline Phosphatase Assay
Both the extract alone and the extract plus glucose-6-P or trehalose-6-P were treated with alkaline phosphatase in the presence or absence of MgCls as follows.
To the dried extracts were added 0.008 mmole of Tris HCl (pH 8.0), 0.02 mmole of MgC12, when present, and 0.1 to 0.2 mg of alkaline phosphatase in a total volume of 1 ml. To determine the amount of trehalose present in the cells before alkaline phosphatase treatment, an identical tube containing extract was incubated at the same time in the absence of the enzyme. Following incubation at 25" for 4.5 hours the reaction was stopped by boiling briefly (approximately 2 min), the samples were either centrifuged and the supernatant solution chromatographed directly, or the samples were extracted with 2 volumes of 95% ethanol, the precipitate was washed, and the supernatant solution and wash were combined and evaporated to dryness. The sample was then dissolved in distilled water and equal aliquots were spotted with and without trehalose controls on Kieselguhr G plates and run in l-butanol-acetone-O.1 M P04, pH 6 (see "Methods"). The intensity of the trehalose region was examined on plates following the detection of sugars by napthoresorcinol spray (9). When MgC& was present in the enzyme assay the samples were desalted prior to chromatography by adsorbing the sugars on 1 g of a 1:2 (w/w) charcoal-Celite mixture and subsequently eluting the sugars with 5$& I-propanol.
The eluates were then evaporated to dryness and treated as above.
Trehalose Synthesis in D. discoideum Vol. 246,No. 17 FIG . 1 (upper left). Identification of trehalose in methanol extracts of young sorocarps (about 22 hours). X-ray film of thin layer plate over which is superimposed (hatched areas) the location of known sugars revealed by spraying the plate with naphthoresorcinol reagent.
The same amount of extract was added at the origin of each strip.
Glucose was added to each (1 to 6). See "Methods" for extraction and thin layer chromatography. FIG. 2 (upper right). Thin layer chromatography of acetates pre-pared from an 80% ethanol extract of young sorocarp (~22 hours). Column 2 is extract alone; S, extract plus maltose octaacetate.
Acetate standards are in Columns 1 and 4. The area just above trehalose in Columns 2 and S did not give the characteristic sugar reaction with naphthoresorcinol spray and hence is not cellobiose. In fact, cellobiose has never been detected in extracts.
FIG . 3, A and B (lower). X-ray films showing the separation of trehalose from other sugars at two stages of differentiation.
The bands are as follows: 1, glucose; 2, maltose or cellobiose or both; 3, trehalose; 4 and 5, unknown. Since the glucose-6-P pool is relatively small and difficult to isolate, it was not feasible to determine the specific activity of glucose-6-P with every experiment.
However, in two experiments performed at late culmination, the specific radioactivity of glucose-6-P was found to be 50% and 66% of the UDP-glucose specific radioactivity.
Therefore, the specific radioactivity of the precursors was routinely taken as 160% of the UDP-glucose specific radioactivity. The rate of trehalose synthesis could then be expressed as an increase with time in micromoles of glucose incorporated per min per ml of packed cells. The per cent of the packed cell volume representing cells (6, 10) at late culmination (20 to 22 hours) was found to be about 50%.

Trehalase and Maltase Assays
Trehalase analyses were carried out in a volume of 0.5 ml containing 15 pmoles of potassium phosphate (pH 6.3), 7.5 pmoles of trehalose, and 0.02 ml of trehalase.
One tube was boiled immediately as a control and the other was incubated for 18 hours at 23" under 1 drop of toluene.
Maltase analyses were carried out in a volume of 2 ml containing 1.25 pmoles of maltose, 8 pmoles of potassium phosphate (pH 6.9), and 4.0 mg of maltase. Again, one sample was boiled immediately and the other was PRECULM, preculmination; CULM, culmination. incubated at 30" for 3 hours.
(A separate maltose control was included.) Glucose produced in these assays was separated from the disaccharides by paper chromatography (descending 24 hours in l-butanol-ethanol-acetone-water, 5:4:3:2, v/v) and the counts per min and micromoles of sugar were determined. Cellobiose was not active as a substrate in these assays.

Other Methods
The phenol-sulfuric acid method of Dubois et al. (11) was used for sugar determinations.
Octaacetates of the sugars were prepared according to the method of Wickberg (12), except that 1 ml each of acetic anhydride and pyridine was used. The acetates were spotted on silica gel plates (13) and run up to 10 cm (two or three times) in 5% ethanol in benzene.
The acetates were detected by spraying the plates with naphthoresorcinol reagent.

RESULTS
Trehalose Identification- Fig.  1 shows the presence of radioactive trehalose and other sugars in an extract prepared after exposure of sorocarp cells to [14C]glucose. Maximum trehalose accumulation occurs in the terminal stages of differentiation, as reported previously (2). Authentic glucose, trehalose, maltose, cellotriose, and cellotetrose were used as standards, and detected by spraying with the napthoresorcinol reagent (hatched areas). Radioactive compounds were detected by x-ray film (dark areas). Since maltose and cellobiose have the same RR value in this solvent system, the extract appears to contain trehalose, maltose CULM, culmination; SORO, sorocarp.