ANALYTICAL
BIOCHEMISTRY
79, 310-318 (1977)
A Radiometric Assay for Trehalose-6Phosphate Synthetase KATHLEEN
A. KILLICK
Boston Biomedical Research Institute, Department of Developmental 20 Staniford Street, Boston, Massachusetts 02114
Biology,
Received August 30, 1976; accepted December 16, 1976 A specific and sensitive radiometric assay has been developed for the measurement of trehalose&phosphate (trehalose-6-P) synthetase activity. With either [*T]glucose-6-P or UDP-[‘*C]glucose as substrate, products unique to the synthetase reaction, i.e., trehalose-6-P and trehalose, may be quantitated after charcoal column chromatography by either high-voltage paper electrophoresis or thin-layer chromatography. Some of the characteristics of this assay for synthetase activity which render it more reliable than calorimetric methods based on nucleoside diphosphate production are: (i) absolute product specificity; no other enzyme besides the synthetase catalyzes the synthesis of trehalose-6-P; (ii) greater sensitivity at low levels of product; and (iii) insensitivity to competing side reactions known to interfere with the calorimetric assay (e.g., polyribonucleotide phosphorylase-catalyzed synthesis or polymerization of nucleoside diphosphates).
Trehalose-6-phosphate (trehalose-6-P) synthetase (UDP-glucose: D-glucose&phosphate I-glucosyltransferase; EC 2.4.1.15) catalyzes the synthesis of trehalose&P, the immediate precursor of the nonreducing disaccharide trehalose from UDP-glucose (1) or GDP-glucose (2-4), and glucose-6-P in several yeasts as well as in higher spore-forming fungi (5,6). Although several assays for the measurement of synthetase activity have been reported, many are less than optimal because of lack of both specificity and sensitivity. During investigations of the regulation of trehalose biosynthesis in the cellular slime mold Dicfyostelium discoideum , it was found that the most sensitive and specific assay of trehalose-6-P synthetase activity was a radiometric procedure in which products unique to the synthetase reaction, i.e., trehalose-6-P and trehalose, were quantitated. The present report describes this procedure for the assay of synthetase activity and its application to enzyme from either undifferentiated myxamoebae or sporulating cells of Dicfyostelium . EXPERIMENTAL
PROCEDURES
Chemicals. [IJ4C]Glucose-6-phosphate (disodium salt), uniformly labeled UDP-[U-14C]glucose, and Aquasol were purchased from New England Nuclear; [U-14C]trehalose was purchased from International Chemical and Nuclear Corporation. The barium salt of trehalose-6310 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN WO3-2697
TREHALOSE-6-P
SYNTHETASE
311
phosphate was synthesized by Mr. Raj Puri. Prior to its use, it was converted to the sodium salt. All other biochemicals were of the highest analytical grade available. Organism and culture conditions. Dictyostelium discoideum [NC-41 ATCC 24697 was grown on nutrient agar with either Escherichia coli or Aerobacter aerogenes as the bacterial associate. Conditions for cell harvesting and for the initiation of development on 2% nonnutrient agar were as previously described (7). Preparation of cell-free extracts. Cells were harvested from nonnutrient agar sheets after either 2 (undifferentiated myxamoebae) or 18 hr (preculmination cells) of starvation in 50 mM MOPS- NaOH (pH 7.5) buffer containing 10 mM potassium phosphate, 5 mM 2-mercaptoethanol, and 25 mM trehalose. Extracts were prepared by subjecting washed cells to a single freeze-thaw, followed by two successive centrifugations (20 min each) at 33,000g. The resultant supernatant liquid served as the source of the synthetase. Assay of trehalose-b-phosphate synthetase activity. Trehalose-6-P synthetase activity was assayed at 23°C in 0.25 ml of an incubation mixture that contained: 25 mM glucose-6-P, 1OmM UDP-glucose, 400 mM KCl, 62.2 mM MgClz, 1 mM EDTA, and 50 mM MES-NaOH (pH 6.5) buffer. In the radiometric assay, the mixture was supplemented with either [ l-14C]glucose-6-P or UDP+*C]glucose. The radioactive substrates had final specific radioactivities of either 199 (preculmination cells) or 800- 1000 cpm/nmol (undifferentiated myxamoebae). The reaction was initiated by addition of enzyme and was terminated by either a 3-min incubation in a boiling water bath or by addition of 2 vol of 95% ethanol. Initial velocities were determined by measuring the rates of synthesis of UDP (calorimetric assay) or of nonreducing disaccharide (radiometric assay). One unit of enzymatic activity is defined as that amount which catalyzes the synthesis of 1 nmol of product/min at 23°C. The specific activity is expressed as units per milligram of acid-precipitable protein. Measurement of UDP. UDP concentrations were measured colorimetritally using the methods of Pontis and Leloir (8). Protein assay. Protein was measured by the method of Lowry et al. (9) using crystalline bovine serum albumin as standard. Measurement of trehalose afterphosphatase treatment. The synthetase assay was terminated by a 3-min incubation in a boiling water bath. After cooling, the contents of each tube were adjusted to pH 8.3 by the addition of Tris-HCI buffer to a final concentration of 100 mM, and carrier trehalose was added to a final level of 2.5 pmol. Trehalase-free alkaline phosphatase (10 ~1; 25.3 unit/mg) was then added, and the mixture was incubated under a drop of toluene overnight at 37°C. The phosphatase reaction was terminated by the addition of 2 vol of 95% ethanol. After 15 min at 23”C, the
312
KATHLEEN
A. KILLICK
solutions were centrifuged (15 min; 3000 rpm). The supe~atant solution was decanted and saved. After the precipitate was washed several times with 70% ethanol, the original supernatant liquid and washings were combined and evaporated to dryness. The residues were dissolved in distilled water, and aliquots were applied to small columns which contained a mixture of 100 mg of acid-washed charcoal and 100 mg of celite. After nonadsorbed material had passed through the column, the charcoal-celite bed was washed with several bed volumes of distilled water. When most of the water had drained from the column, the latter was washed with approximately 15 ml of I-propanol(5%). The alcohol fractions were then lyophilized to dryness and redissolved in distilled water. Thin-layer plates (20 x 20 cm) were prepared (four plates at a time) from a slurry of 30 g of Kieselguhr G in 60 ml of pH 6.0 potassium phosphate buffer (100 mM) to a thickness of 250 nm (10). After being spread, the plates were allowed to air-dry overnight. Aliquots of sugar solution (previously desalted on charcoal) were spotted onto thin-layer plates, 2 cm from the edge of the plate, and were run in ascending chromatography in l-butanol-acetone-O. 1 M phosphate (4:5: 1, v/v) at pH 6.0 to 16 cm above the origin. Each plate was run twice in the solvent, with drying between runs. The dried plates were then scanned for radioactivity using the following methods. Starting at approximately 1 cm below the origin and moving toward the top (or “front”) of the plate, each column to which radioactive sample had been applied was completely sectioned (to the solvent front) into 2.5-mm segments with a razor blade. Each section was scraped into a scintillation vial and incubated with 1 ml of distilled water overnight. Aliquots of eluate were mixed with Aquasol (10 ml), and the radioactivity was measured in a liquid scintillation spectrometer. The amount of [14C]trehalose was calculated from the total radioactivity in that peak having the same R, as authentic standard trehalose and from the specific radioactivity of either the [14C]glucose-6-P or UDP-[14C]glucose used as substrate. If the radioactivity associated with the trehalose area was not resolvable as a single sharp peak, the respective eluates were pooled and evaporated to dryness. After being desalted on charcoal, aliquots were resubjected to tic as described before, Measurement of trehalose in absence of phosphatase treatment. The synthetase assay was te~inated by addition of 2 vol of 95% ethanol, and carrier trehalose was added to a final level of 2.5 pmol. After 15 min at 23”C, the solutions were centrifuged (15 min; 3000 rpm). The supernatant solution was decanted and saved. After the precipitate was washed several times with 70% ethanol, the supernatant liquid and washings were pooled and evaporated to dryness. Desalting on charcoal and the methods used for thin-layer chromatography were the same as described before.
TREHALOSE-6-P
SYNTHETASE
313
Measurement of Trehalose-6-P. The synthetase assay was terminated by the addition of 2 vol of 95% ethanol, and 2.5 ,umol of carrier trehalose-6-P was added. After 15 min at 23”C, insoluble material was removed by centrifugation. The precipitate was washed several times with 70% ethanol, and the supematant liquid and washings were pooled and evaporated to dryness. The residues were solubilized in distilled water, and aliquots were applied to charcoal-celite columns as described before. The solutions were allowed to drain through the columns under gravity. After most of the applied liquid had eluted from the column, the latter was washed with lo-15 ml of 1-propanol (5%). The alcohol fractions (containing both trehalose and trehalose-6-P) were lyophilized to dryness and redissolved in distilled water. Aliquots were spotted onto Whatman No. 3 MM chromatography paper and were subjected to high-voltage paper electrophoresis in either 50 mM sodium tetraborate (pH 9.7) (1) or 0.2 M ammonium formate buffer (pH 3.6), (4) in an E. C. Apparatus Co. pressure-plate electrophoresis apparatus. The [14C]trehalose-6-P area was identified by its rate of migration compared to that of standard trehalose-6-P run under identical conditions on a companion strip and subsequently visualized with silver nitrate. After electrophoresis, the electrophoretograms were dried and then scanned for radioactivity using the following methods. Starting at approximately 2 cm below the origin and moving toward the anode end of the paper, each strip was cut into 2.5-mm segments (60-70 in total). After elution of each segment with either distilled water or 5 mM acetic acid, aliquots of the resulting solutions were mixed with Aquasol, and the radioactivity was measured in a liquid scintillation spectrometer. The amount of [14C]trehalose-6-P was calculated from the total radioactivity in that peak having the same R.,, as authentic standard trehalose-6-P and from the specific radioactivity of the [14C]glucose-6-P used as substrate. When the radioactivity associated with the trehalose-6-P area was not resolvable as a single sharp peak, the respective eluates were pooled, concentrated, and resubmitted to electrophoresis. RESULTS Measurement
AND DISCUSSION
of Products
The two immediate products of the trehalose-6-P synthetase reaction are trehalose-6-P and UDP. Since extracts prepared from both undifferentiated myxamoebae and early sorocarps contained phosphatase activity, some of the trehalose-6-P produced during the synthetase assay was subsequently hydrolyzed to trehalose. Thus, assessment of synthetase activity in terms of total disaccharide product demanded that either both trehalose-6-P and trehalose be measured or that total trehalose be determined after phosphatase treatment of the boiled synthetase mixture.
314
KATHLEEN
A. KILLICK
In those cases where total nonreducing disaccharide is measured (after phosphatase treatment), either thin-layer chromatography (tic) (Fig. 1) or high-voltage paper electrophoresis may be used (Fig. 2). The results depicted in Fig. 1 show a typical scan after tic of the synthetase products, using UDP-[14C]glucose as substrate. The [14C]trehalose area of this chromatogram was identified by its rate of migration relative to that of authentic standard trehalose run on a companion strip. Control experiments demonstrated that, when the radioactivity in this section was eluted from the plate and subsequently incubated with a specific trehalase purified from Dictyostelium, at least 75% of the radioactivity exhibited the same mobility as authentic glucose when determined with tic. Although laborious, the use of charcoal columns had an advantage in that most of the labeled substrate (i.e., UDP-[14C]glucose) and all of the product (i.e., trehalose) were adsorbed onto the charcoal, whereas only the disaccharide was eluted with 1-propanol (5%). Moreover, when [14C]glucose-6-P was used as labeled substrate in place of UDP[14C]glucose, all of the [14C]glucose resulting after phosphatase treatment I
I
I
I
so FROM
80
I
1
TREHALOSE
0
20
40
DISTANCE
ORIGIN
I
I
loo
120
(mm
1
)
FIG. 1. Thin-layer chromatography of trehalose-6-P synthetase reaction products. Extracts were prepared from 1%hr cells in MPMT buffer, and enzyme (0.155 mg of protein) was incubated for 60 min (23°C) with all assay constituents plus LJDP-[T]glucose (152 cpm/nmol). Chromatogmphy was carried out as described in Experimental Procedures.
TREHALOSE-6-P I
I
315
SYNTHETASE
I
I
I
I
w,ooo
a! 3
1
lop00
B : a 6,000 c,
2,000 d
1 0 DISTANCE
FROH
OR/O/N
(mm1
FIG. 2. High-voltage paper electrophoresis of trehalose-6-P synthetase reaction products. Extracts were prepared from 18-hr cells in MPMT buffer, and enzyme (1 mg of protein) was incubated 60 min (23°C) with all assay ~onsti~ents plus p4C]glucose-f$P (199 cpm/nmol). Electrophoresis was carried out with 50 mM sodium tetraborate buffer (pH 9.7) at 600 V.
eluted from the column in the first water wash, leaving the disaccharide bound to the charcoal. Hence, by the combination of charcoal column and thin-layer chromatography, separation of substrate and end product was readily effected. With these methods, the recovery of known amounts of [l*C]trehalose was 7545%. An alternative method for measuring both trehalose-6-P and trehalose involved the use of high-voltage paper electrophoresis. When this method was used for the assay of trehalose-6-P synthetase, [‘*C]glucose-6-P was used as a substrate, and the phosphat~e step was omitted. Figure 2 shows a typical scan after paper electrophoresis of the synthetase products in 50 mM borate buffer (pH 9.7). Charcoal columns were used to desalt samples prior to electrophoresis (see Experimental Procedure). The columns used in these experiments were not, however, in contrast to those used prior to tic, washed extensively with water prior to saccharide elution with propanol. This additional washing was not essential to subsequent resolution (see Fig. 2) and merely decreased the recovery of [14C]trehalose-6-P. It is apparent from the scan shown in Fig. 2 that the aforementioned methodology resulted in the resolution of four major peaks of radioactivity. Both the glucose and glucose-6-P areas were identified by their rates of migration relative to those of standard compounds. The ~l~C]~eh~ose~P
316
KATHLEEN
A. KILLICK
area was identified by its rate of migration compared to that of the standard trehalose-6-P run under identical conditions on a companion strip and subsequently visualized with silver nitrate. Control experiments showed that, when the radioactivity in this area was eluted from the paper and then incubated with bacterial alkaline phosphatase (trehalose-free), 78-82% of the radioactivity exhibited the same mobility as authentic trehalose as determined with thin-layer chromatography. Most of the radioactivity which remained at the origin, as shown in Fig. 2, was due to [14C]trehalose. This was confirmed by eluting the radioactivity, desalting, and subsequently subjecting aliquots of the solution to tic (see Experimental Procedure). In addition to the identification, isolation, and subsequent quantitation of both trehalose-6-P and trehalose, trehalose-6-P synthetase activity may be assayed by measuring the rate of UDP synthesis (see Experimental Procedure). Although this is, by far, the most convenient method, it lacks the sensitivity and absolute product specificity associated with the radiometric procedure. Assay of Trehalose-6-P
Synthetase from Sporulation
Cells
Extracts were prepared during early sporulation (i.e., preculmination), and trehalose-6-P synthetase activity was measured by following the rates of synthesis of both UDP and nonreducing disaccharide (i.e., trehalose-6-P plus trehalose). Excellent stoichiometry was observed between the nucleoside diphosphate and disaccharide products. Hence, with these extracts, either the calorimetric (8) or the radiometric assay gave a valid assessment of synthetase activity. In both cases, the specific activity was 40 unit/mg. One advantage to the use of the radiometric assay not shared by the calorimetric procedure was that, with the former method, since the rates of trehalose-6-P and trehalose were both measured, it was possible to determine the extent to which phosphatase activity was limiting during the synthetase assay (Fig. 3). Assay of Trehalose-4-P
Synthetase from
Undifferentiated
Myxamoebae
Attempts to assay synthetase activity inmyxamoebae extracts (prepared as described in Experimental Procedure) using the calorimetric procedure (8) were unsuccessful. Nucleoside diphosphate produc.tion was neither linear with time nor stoichiomettic relative to disaccharide synthesis. The absence of stoichiometry may have been due at least, in part, to the fact that the above extracts contained a significant amount of polyribonucleotide phosphorylase (PNPase) activity (either of bacterial or slime mold origin) (11). Since the synthetase was prepared in the presence of 10 mM phosphate and was subsequently assayed in a 3.4 mM solution of this ion, it
TREHALOSE-6-P r
,
i
t
MYXAMQEBAE
2
e:
300
-
2 6
200
-
A TOP /
a0 %
/
l 100 O&A 0
/
*
t
--
30
60 M/#UTES
I
so
120
1
I
I
FRECULMINATION
6
1600 ‘1: * % c
T6P
,/
1200-
600
317
SYNTHETASE
-
l
/ HT
/
0
30
/*
60
1 so
t 120
FIG. 3. Trehalose-6-P synthetase activity in myxamoebae and spore-forming cells. Extracts were prepared in MPMT buffer from undifferentiated cells (A) and those engaged in sporulation (B) and were assayed for synthetase activity using ~14C]g1ucose-6-P at either a specific radioactivity of 1000 (A) or 199 cpmlnmol (B). The amount of protein used in both cases was 0.5 mg. The concentration of carrier trehalose during the assay was 5 mM, that of phosphate was 3.4 mM.
is quite possible that PNPase catalyzed endogenous phosphoryolytic reactions, which subsequently interfered with the calorimetric assay of synthetase activity. Because of these considerations, it was apparent that the radiometric assay was the only reliable method for assaying synthetase activity from undifferentiated cells. The results of applying this assay are shown in Fig. 3. Myxamoebae synthetase was assayed (see Experimental Procedure) by measu~ng the rates of trehalose-6-P and trehalose synthesis using high-voltage paper electrophoresis and tic, respectively, for quantitating the two products unique to the synthetase reaction, It is apparent that phosphatase activity in these extracts was more limiting (Fig. 3A) than it was in extracts from sporulating cells (Fig. 3B). Also apparent is the fact that activation of myxamoebae synthetase occurred after an initial lag period. Since, with all preparations of synthetase studied (using extracts
318
KATHLEEN
A. KILLICK
from both E. coli- and A. aerogenes-grown cells), the rate of trehalose production appeared to be constant with time, while that of trehalose-6-P increased, it is likely that trehalose-6-P synthetase (as opposed to a phosphatase) was activated. The final specific activity for the example cited in Fig. 3A was 6.6 unit/mg. ACKNOWLEDGMENT This investigation was supported by Research Grants AGO0433 and AGO0260 to Dr. B. E. Wright from the National Institutes of Health.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Cabib, E., and Leloir, L. F. (1958) J. Biol. C&m. 231, 259-275. Elbein, A. D. (1967) J. Biol. Chem. 242, 403-406. Elbein, A. D. (1%8) J. Bacterial. 96, 1623-1631. Liu, C., Patterson, B. W., Lapp, D., and Elbein, A. D. (1969) J. Biol. Chem. 244, 3728-3731. Elbein, A. D. (1974) Advnn. Carbohyd. Chem. Biochem. 30, 227-256. Wright, B. E., and Killick, K. A. (1975) in Spores (Costilow, R. N., Sadoff, H. L., and Gerhardt, P., eds.), Vol. 6, pp. 73-84, American Society of Microbiologists, Bethesda, Maryland. Liddel, G. U., and Wright, B. E. (1961) Develop. Biol. 3, 265-276. Pontis. H. Cl., and Leloir, L. F. (1962) Methods Biochem. Anal. 10, 107- 136. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Waldi, D. (1%5) J. Chromatogr. 18, 417-418. Killick, K. A. (1976) Fed. Proc. 35, 1399.
BIOCHEMISTRY
79, 310-318 (1977)
A Radiometric Assay for Trehalose-6Phosphate Synthetase KATHLEEN
A. KILLICK
Boston Biomedical Research Institute, Department of Developmental 20 Staniford Street, Boston, Massachusetts 02114
Biology,
Received August 30, 1976; accepted December 16, 1976 A specific and sensitive radiometric assay has been developed for the measurement of trehalose&phosphate (trehalose-6-P) synthetase activity. With either [*T]glucose-6-P or UDP-[‘*C]glucose as substrate, products unique to the synthetase reaction, i.e., trehalose-6-P and trehalose, may be quantitated after charcoal column chromatography by either high-voltage paper electrophoresis or thin-layer chromatography. Some of the characteristics of this assay for synthetase activity which render it more reliable than calorimetric methods based on nucleoside diphosphate production are: (i) absolute product specificity; no other enzyme besides the synthetase catalyzes the synthesis of trehalose-6-P; (ii) greater sensitivity at low levels of product; and (iii) insensitivity to competing side reactions known to interfere with the calorimetric assay (e.g., polyribonucleotide phosphorylase-catalyzed synthesis or polymerization of nucleoside diphosphates).
Trehalose-6-phosphate (trehalose-6-P) synthetase (UDP-glucose: D-glucose&phosphate I-glucosyltransferase; EC 2.4.1.15) catalyzes the synthesis of trehalose&P, the immediate precursor of the nonreducing disaccharide trehalose from UDP-glucose (1) or GDP-glucose (2-4), and glucose-6-P in several yeasts as well as in higher spore-forming fungi (5,6). Although several assays for the measurement of synthetase activity have been reported, many are less than optimal because of lack of both specificity and sensitivity. During investigations of the regulation of trehalose biosynthesis in the cellular slime mold Dicfyostelium discoideum , it was found that the most sensitive and specific assay of trehalose-6-P synthetase activity was a radiometric procedure in which products unique to the synthetase reaction, i.e., trehalose-6-P and trehalose, were quantitated. The present report describes this procedure for the assay of synthetase activity and its application to enzyme from either undifferentiated myxamoebae or sporulating cells of Dicfyostelium . EXPERIMENTAL
PROCEDURES
Chemicals. [IJ4C]Glucose-6-phosphate (disodium salt), uniformly labeled UDP-[U-14C]glucose, and Aquasol were purchased from New England Nuclear; [U-14C]trehalose was purchased from International Chemical and Nuclear Corporation. The barium salt of trehalose-6310 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN WO3-2697
TREHALOSE-6-P
SYNTHETASE
311
phosphate was synthesized by Mr. Raj Puri. Prior to its use, it was converted to the sodium salt. All other biochemicals were of the highest analytical grade available. Organism and culture conditions. Dictyostelium discoideum [NC-41 ATCC 24697 was grown on nutrient agar with either Escherichia coli or Aerobacter aerogenes as the bacterial associate. Conditions for cell harvesting and for the initiation of development on 2% nonnutrient agar were as previously described (7). Preparation of cell-free extracts. Cells were harvested from nonnutrient agar sheets after either 2 (undifferentiated myxamoebae) or 18 hr (preculmination cells) of starvation in 50 mM MOPS- NaOH (pH 7.5) buffer containing 10 mM potassium phosphate, 5 mM 2-mercaptoethanol, and 25 mM trehalose. Extracts were prepared by subjecting washed cells to a single freeze-thaw, followed by two successive centrifugations (20 min each) at 33,000g. The resultant supernatant liquid served as the source of the synthetase. Assay of trehalose-b-phosphate synthetase activity. Trehalose-6-P synthetase activity was assayed at 23°C in 0.25 ml of an incubation mixture that contained: 25 mM glucose-6-P, 1OmM UDP-glucose, 400 mM KCl, 62.2 mM MgClz, 1 mM EDTA, and 50 mM MES-NaOH (pH 6.5) buffer. In the radiometric assay, the mixture was supplemented with either [ l-14C]glucose-6-P or UDP+*C]glucose. The radioactive substrates had final specific radioactivities of either 199 (preculmination cells) or 800- 1000 cpm/nmol (undifferentiated myxamoebae). The reaction was initiated by addition of enzyme and was terminated by either a 3-min incubation in a boiling water bath or by addition of 2 vol of 95% ethanol. Initial velocities were determined by measuring the rates of synthesis of UDP (calorimetric assay) or of nonreducing disaccharide (radiometric assay). One unit of enzymatic activity is defined as that amount which catalyzes the synthesis of 1 nmol of product/min at 23°C. The specific activity is expressed as units per milligram of acid-precipitable protein. Measurement of UDP. UDP concentrations were measured colorimetritally using the methods of Pontis and Leloir (8). Protein assay. Protein was measured by the method of Lowry et al. (9) using crystalline bovine serum albumin as standard. Measurement of trehalose afterphosphatase treatment. The synthetase assay was terminated by a 3-min incubation in a boiling water bath. After cooling, the contents of each tube were adjusted to pH 8.3 by the addition of Tris-HCI buffer to a final concentration of 100 mM, and carrier trehalose was added to a final level of 2.5 pmol. Trehalase-free alkaline phosphatase (10 ~1; 25.3 unit/mg) was then added, and the mixture was incubated under a drop of toluene overnight at 37°C. The phosphatase reaction was terminated by the addition of 2 vol of 95% ethanol. After 15 min at 23”C, the
312
KATHLEEN
A. KILLICK
solutions were centrifuged (15 min; 3000 rpm). The supe~atant solution was decanted and saved. After the precipitate was washed several times with 70% ethanol, the original supernatant liquid and washings were combined and evaporated to dryness. The residues were dissolved in distilled water, and aliquots were applied to small columns which contained a mixture of 100 mg of acid-washed charcoal and 100 mg of celite. After nonadsorbed material had passed through the column, the charcoal-celite bed was washed with several bed volumes of distilled water. When most of the water had drained from the column, the latter was washed with approximately 15 ml of I-propanol(5%). The alcohol fractions were then lyophilized to dryness and redissolved in distilled water. Thin-layer plates (20 x 20 cm) were prepared (four plates at a time) from a slurry of 30 g of Kieselguhr G in 60 ml of pH 6.0 potassium phosphate buffer (100 mM) to a thickness of 250 nm (10). After being spread, the plates were allowed to air-dry overnight. Aliquots of sugar solution (previously desalted on charcoal) were spotted onto thin-layer plates, 2 cm from the edge of the plate, and were run in ascending chromatography in l-butanol-acetone-O. 1 M phosphate (4:5: 1, v/v) at pH 6.0 to 16 cm above the origin. Each plate was run twice in the solvent, with drying between runs. The dried plates were then scanned for radioactivity using the following methods. Starting at approximately 1 cm below the origin and moving toward the top (or “front”) of the plate, each column to which radioactive sample had been applied was completely sectioned (to the solvent front) into 2.5-mm segments with a razor blade. Each section was scraped into a scintillation vial and incubated with 1 ml of distilled water overnight. Aliquots of eluate were mixed with Aquasol (10 ml), and the radioactivity was measured in a liquid scintillation spectrometer. The amount of [14C]trehalose was calculated from the total radioactivity in that peak having the same R, as authentic standard trehalose and from the specific radioactivity of either the [14C]glucose-6-P or UDP-[14C]glucose used as substrate. If the radioactivity associated with the trehalose area was not resolvable as a single sharp peak, the respective eluates were pooled and evaporated to dryness. After being desalted on charcoal, aliquots were resubjected to tic as described before, Measurement of trehalose in absence of phosphatase treatment. The synthetase assay was te~inated by addition of 2 vol of 95% ethanol, and carrier trehalose was added to a final level of 2.5 pmol. After 15 min at 23”C, the solutions were centrifuged (15 min; 3000 rpm). The supernatant solution was decanted and saved. After the precipitate was washed several times with 70% ethanol, the supernatant liquid and washings were pooled and evaporated to dryness. Desalting on charcoal and the methods used for thin-layer chromatography were the same as described before.
TREHALOSE-6-P
SYNTHETASE
313
Measurement of Trehalose-6-P. The synthetase assay was terminated by the addition of 2 vol of 95% ethanol, and 2.5 ,umol of carrier trehalose-6-P was added. After 15 min at 23”C, insoluble material was removed by centrifugation. The precipitate was washed several times with 70% ethanol, and the supematant liquid and washings were pooled and evaporated to dryness. The residues were solubilized in distilled water, and aliquots were applied to charcoal-celite columns as described before. The solutions were allowed to drain through the columns under gravity. After most of the applied liquid had eluted from the column, the latter was washed with lo-15 ml of 1-propanol (5%). The alcohol fractions (containing both trehalose and trehalose-6-P) were lyophilized to dryness and redissolved in distilled water. Aliquots were spotted onto Whatman No. 3 MM chromatography paper and were subjected to high-voltage paper electrophoresis in either 50 mM sodium tetraborate (pH 9.7) (1) or 0.2 M ammonium formate buffer (pH 3.6), (4) in an E. C. Apparatus Co. pressure-plate electrophoresis apparatus. The [14C]trehalose-6-P area was identified by its rate of migration compared to that of standard trehalose-6-P run under identical conditions on a companion strip and subsequently visualized with silver nitrate. After electrophoresis, the electrophoretograms were dried and then scanned for radioactivity using the following methods. Starting at approximately 2 cm below the origin and moving toward the anode end of the paper, each strip was cut into 2.5-mm segments (60-70 in total). After elution of each segment with either distilled water or 5 mM acetic acid, aliquots of the resulting solutions were mixed with Aquasol, and the radioactivity was measured in a liquid scintillation spectrometer. The amount of [14C]trehalose-6-P was calculated from the total radioactivity in that peak having the same R.,, as authentic standard trehalose-6-P and from the specific radioactivity of the [14C]glucose-6-P used as substrate. When the radioactivity associated with the trehalose-6-P area was not resolvable as a single sharp peak, the respective eluates were pooled, concentrated, and resubmitted to electrophoresis. RESULTS Measurement
AND DISCUSSION
of Products
The two immediate products of the trehalose-6-P synthetase reaction are trehalose-6-P and UDP. Since extracts prepared from both undifferentiated myxamoebae and early sorocarps contained phosphatase activity, some of the trehalose-6-P produced during the synthetase assay was subsequently hydrolyzed to trehalose. Thus, assessment of synthetase activity in terms of total disaccharide product demanded that either both trehalose-6-P and trehalose be measured or that total trehalose be determined after phosphatase treatment of the boiled synthetase mixture.
314
KATHLEEN
A. KILLICK
In those cases where total nonreducing disaccharide is measured (after phosphatase treatment), either thin-layer chromatography (tic) (Fig. 1) or high-voltage paper electrophoresis may be used (Fig. 2). The results depicted in Fig. 1 show a typical scan after tic of the synthetase products, using UDP-[14C]glucose as substrate. The [14C]trehalose area of this chromatogram was identified by its rate of migration relative to that of authentic standard trehalose run on a companion strip. Control experiments demonstrated that, when the radioactivity in this section was eluted from the plate and subsequently incubated with a specific trehalase purified from Dictyostelium, at least 75% of the radioactivity exhibited the same mobility as authentic glucose when determined with tic. Although laborious, the use of charcoal columns had an advantage in that most of the labeled substrate (i.e., UDP-[14C]glucose) and all of the product (i.e., trehalose) were adsorbed onto the charcoal, whereas only the disaccharide was eluted with 1-propanol (5%). Moreover, when [14C]glucose-6-P was used as labeled substrate in place of UDP[14C]glucose, all of the [14C]glucose resulting after phosphatase treatment I
I
I
I
so FROM
80
I
1
TREHALOSE
0
20
40
DISTANCE
ORIGIN
I
I
loo
120
(mm
1
)
FIG. 1. Thin-layer chromatography of trehalose-6-P synthetase reaction products. Extracts were prepared from 1%hr cells in MPMT buffer, and enzyme (0.155 mg of protein) was incubated for 60 min (23°C) with all assay constituents plus LJDP-[T]glucose (152 cpm/nmol). Chromatogmphy was carried out as described in Experimental Procedures.
TREHALOSE-6-P I
I
315
SYNTHETASE
I
I
I
I
w,ooo
a! 3
1
lop00
B : a 6,000 c,
2,000 d
1 0 DISTANCE
FROH
OR/O/N
(mm1
FIG. 2. High-voltage paper electrophoresis of trehalose-6-P synthetase reaction products. Extracts were prepared from 18-hr cells in MPMT buffer, and enzyme (1 mg of protein) was incubated 60 min (23°C) with all assay ~onsti~ents plus p4C]glucose-f$P (199 cpm/nmol). Electrophoresis was carried out with 50 mM sodium tetraborate buffer (pH 9.7) at 600 V.
eluted from the column in the first water wash, leaving the disaccharide bound to the charcoal. Hence, by the combination of charcoal column and thin-layer chromatography, separation of substrate and end product was readily effected. With these methods, the recovery of known amounts of [l*C]trehalose was 7545%. An alternative method for measuring both trehalose-6-P and trehalose involved the use of high-voltage paper electrophoresis. When this method was used for the assay of trehalose-6-P synthetase, [‘*C]glucose-6-P was used as a substrate, and the phosphat~e step was omitted. Figure 2 shows a typical scan after paper electrophoresis of the synthetase products in 50 mM borate buffer (pH 9.7). Charcoal columns were used to desalt samples prior to electrophoresis (see Experimental Procedure). The columns used in these experiments were not, however, in contrast to those used prior to tic, washed extensively with water prior to saccharide elution with propanol. This additional washing was not essential to subsequent resolution (see Fig. 2) and merely decreased the recovery of [14C]trehalose-6-P. It is apparent from the scan shown in Fig. 2 that the aforementioned methodology resulted in the resolution of four major peaks of radioactivity. Both the glucose and glucose-6-P areas were identified by their rates of migration relative to those of standard compounds. The ~l~C]~eh~ose~P
316
KATHLEEN
A. KILLICK
area was identified by its rate of migration compared to that of the standard trehalose-6-P run under identical conditions on a companion strip and subsequently visualized with silver nitrate. Control experiments showed that, when the radioactivity in this area was eluted from the paper and then incubated with bacterial alkaline phosphatase (trehalose-free), 78-82% of the radioactivity exhibited the same mobility as authentic trehalose as determined with thin-layer chromatography. Most of the radioactivity which remained at the origin, as shown in Fig. 2, was due to [14C]trehalose. This was confirmed by eluting the radioactivity, desalting, and subsequently subjecting aliquots of the solution to tic (see Experimental Procedure). In addition to the identification, isolation, and subsequent quantitation of both trehalose-6-P and trehalose, trehalose-6-P synthetase activity may be assayed by measuring the rate of UDP synthesis (see Experimental Procedure). Although this is, by far, the most convenient method, it lacks the sensitivity and absolute product specificity associated with the radiometric procedure. Assay of Trehalose-6-P
Synthetase from Sporulation
Cells
Extracts were prepared during early sporulation (i.e., preculmination), and trehalose-6-P synthetase activity was measured by following the rates of synthesis of both UDP and nonreducing disaccharide (i.e., trehalose-6-P plus trehalose). Excellent stoichiometry was observed between the nucleoside diphosphate and disaccharide products. Hence, with these extracts, either the calorimetric (8) or the radiometric assay gave a valid assessment of synthetase activity. In both cases, the specific activity was 40 unit/mg. One advantage to the use of the radiometric assay not shared by the calorimetric procedure was that, with the former method, since the rates of trehalose-6-P and trehalose were both measured, it was possible to determine the extent to which phosphatase activity was limiting during the synthetase assay (Fig. 3). Assay of Trehalose-4-P
Synthetase from
Undifferentiated
Myxamoebae
Attempts to assay synthetase activity inmyxamoebae extracts (prepared as described in Experimental Procedure) using the calorimetric procedure (8) were unsuccessful. Nucleoside diphosphate produc.tion was neither linear with time nor stoichiomettic relative to disaccharide synthesis. The absence of stoichiometry may have been due at least, in part, to the fact that the above extracts contained a significant amount of polyribonucleotide phosphorylase (PNPase) activity (either of bacterial or slime mold origin) (11). Since the synthetase was prepared in the presence of 10 mM phosphate and was subsequently assayed in a 3.4 mM solution of this ion, it
TREHALOSE-6-P r
,
i
t
MYXAMQEBAE
2
e:
300
-
2 6
200
-
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a0 %
/
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/
*
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--
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60 M/#UTES
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so
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1
I
I
FRECULMINATION
6
1600 ‘1: * % c
T6P
,/
1200-
600
317
SYNTHETASE
-
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/ HT
/
0
30
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t 120
FIG. 3. Trehalose-6-P synthetase activity in myxamoebae and spore-forming cells. Extracts were prepared in MPMT buffer from undifferentiated cells (A) and those engaged in sporulation (B) and were assayed for synthetase activity using ~14C]g1ucose-6-P at either a specific radioactivity of 1000 (A) or 199 cpmlnmol (B). The amount of protein used in both cases was 0.5 mg. The concentration of carrier trehalose during the assay was 5 mM, that of phosphate was 3.4 mM.
is quite possible that PNPase catalyzed endogenous phosphoryolytic reactions, which subsequently interfered with the calorimetric assay of synthetase activity. Because of these considerations, it was apparent that the radiometric assay was the only reliable method for assaying synthetase activity from undifferentiated cells. The results of applying this assay are shown in Fig. 3. Myxamoebae synthetase was assayed (see Experimental Procedure) by measu~ng the rates of trehalose-6-P and trehalose synthesis using high-voltage paper electrophoresis and tic, respectively, for quantitating the two products unique to the synthetase reaction, It is apparent that phosphatase activity in these extracts was more limiting (Fig. 3A) than it was in extracts from sporulating cells (Fig. 3B). Also apparent is the fact that activation of myxamoebae synthetase occurred after an initial lag period. Since, with all preparations of synthetase studied (using extracts
318
KATHLEEN
A. KILLICK
from both E. coli- and A. aerogenes-grown cells), the rate of trehalose production appeared to be constant with time, while that of trehalose-6-P increased, it is likely that trehalose-6-P synthetase (as opposed to a phosphatase) was activated. The final specific activity for the example cited in Fig. 3A was 6.6 unit/mg. ACKNOWLEDGMENT This investigation was supported by Research Grants AGO0433 and AGO0260 to Dr. B. E. Wright from the National Institutes of Health.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Cabib, E., and Leloir, L. F. (1958) J. Biol. C&m. 231, 259-275. Elbein, A. D. (1967) J. Biol. Chem. 242, 403-406. Elbein, A. D. (1%8) J. Bacterial. 96, 1623-1631. Liu, C., Patterson, B. W., Lapp, D., and Elbein, A. D. (1969) J. Biol. Chem. 244, 3728-3731. Elbein, A. D. (1974) Advnn. Carbohyd. Chem. Biochem. 30, 227-256. Wright, B. E., and Killick, K. A. (1975) in Spores (Costilow, R. N., Sadoff, H. L., and Gerhardt, P., eds.), Vol. 6, pp. 73-84, American Society of Microbiologists, Bethesda, Maryland. Liddel, G. U., and Wright, B. E. (1961) Develop. Biol. 3, 265-276. Pontis. H. Cl., and Leloir, L. F. (1962) Methods Biochem. Anal. 10, 107- 136. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Waldi, D. (1%5) J. Chromatogr. 18, 417-418. Killick, K. A. (1976) Fed. Proc. 35, 1399.
