ANALYTICAL
BIOCHEMISTRY
Synthesis
49,
!%?-2%
of Glucose
Labeled BRUCE
(1972)
1 -Phosphate-U-l%
Sucroses
Using
111. CHASSY
Env~r~~rne~tal ~e~ha~~rns ~~at~onal ~~st~tut~s
and
Sucrose MICAH
AND
Specifically
Phospho~lase I. KRICHEVSKY
Section, ~~~~o~al of
Health
Institute of Dental Research, Bethesda, Maryland SOOl~
Received February
9, 1972
More than twenty years ago Hassid, Doudoroff, and Barker investigated the mechanism of action of sucrose phosphorylase (disaccharide glucosyltransferase (non-specific), EC 2.4.1.7) isolated from Pseudomonas sacc~a~o~~~~ using radioisotopes (l-3). These classical studies not only led to the postulation of the following mechanism: (1) sucrose + enzyme -+ glucosyl-enzyme (2) glu~syl-enzyme
+ X(X
+ fructose,
= l?04, A,O+ or OH-) -+ glucose-l-X
+ enzyme
but also led to the synthesis of specifically labeled sucroses (e.g., sucrose labeled with 14C in either the glucose or the fructose moiety (4,4a)). Abeles and his coworkers have elucidated more of the details of the mechanism of action of sucrose l~hosphorylase and added some modern methodology to the synthesis of labeled sucroses (5,6). Current investigations of the physiology of oral streptococci (7,8) created a need for a method of synthesizing specifically labeled sueroses of high purity and specific activity by a straightforward and economic route. This report (1) growth of Leuconostoc mesenteroides presents methods for the: (ATCC 12291), (2) preparation of sucrose phosphorylase in high yields, (5) preparation of glucose l-phosphate-U-l~~ (and fructose-U-l*C as a side product), (4) preparation of sucrose labeled with 14C in either the fructose or the glucose moiety, and (5) analysis of the isotopic purity of the products. PROCEDURES
Growth of L. mesenteroides P-39 (ATCC 12291) (9) The composition of the medium used for growth of L. mesentero~s is, in gm/liter: Tryptone (Difco) 40, yeast extract (Difco) 25, K,HPO, 50, sucrose 40, and 0.25 ml/liter of a vitamin-salts solution containing, in gmlliter: thiamine-HCI 1, ascorbic acid 10, and MgSO,*7H,O 40, 232
@ 1972 by Academic Press, Inc. All righb of reproduction in any form reserved.
SYNTHESIS
OF
SPECIFICALLY
LABELED
SUCROSE
233
FeS04.7H,0 1, and MnS04.Hz0 20. The medium was sterilized at 121” for 1 hr (in 20 liter carboys). The two 20 liter carboys were inoculated with 200 ml each of a culture actively growing in the same medium. After 23 hr at 31”, the bacteria were harvested by continuous centrifugation (DeLaval cream separator, Poughkeepsie, N. Y.). The cells were washed three times with cold 0.033 M potassium phosphate buffer, pH 6.8, and collected each time by centrifugation at 12,000g for 15 min. The yield was 58 gm of packed white cells, which were frozen for future use. Enzyme
Assays
Phosphate uptake method. This assay procedure measures the amount of sucrose phosphorolysis by Fiske and SubbaRow (10) determination of phosphate disappearance. A unit is defined as 1 pmole of Pi incorporated in 20 min at 30” in 0.033 M phosphate containing 0.1 M sucrose at pH 6.64. Pyridine nucleotide linked assay. This assay is based on the following reaction pathway: sllc*ose
phosphorylase
sucrose
+ Pi
’ G-l-P
+ fruct’ose
(1)
+ ADP
(3
hexokinase
fructose
+ ATP-
F-6-P hexose-phosphate isomerase
F-6-P
,
’ G-6-P
(3,
G-6-P dehydrogenase
G-6-P
+ XAD
’ 6-PGA
+ NADII
+ H’
14‘)
Hexose assay methods based on hexokinase and glucose-6-phosphate dehydrogenase are commercially available (Calbiochem, Stat-Pack, San Diego, Calif.). The solutions are prepared at double strength, 0.5 ml aliquots are adjusted to 1 ml with water, sucrose (0.1 M final concentration), phosphate buffer pH 7.4 (0.1 M final concentration), 5 IU (2 ~1) of an ammonium sulfate suspension of crystalline phosphohexose isomerase, and the enzyme solution to be tested. The assay is useful from a concentrat,ion of 0.005 to 0.1 unit of enzyme per milliliter. Purification of sucrose phosphorylase. 15 gm of frozen L. mesenteroides cells was thawed and slurried with 10 ml of 0.033 M potassium phosphate buffer, pH 6.8. The thick suspension was passed 3 times through a French Pressure cell (10,000 psi, Aminco, Silver Spring, Md.). The pressed preparation was cent,rifuged at 27,000g for 15 min. The supernatant (26 ml) was withdrawn with a pipet and 26 ml of cold saturated ammonium sulfate was added slowly at 0” with constant stirring. The
234
CHASSY
AND
KRICHEVSKY
solution was stirred for an additional 15 min and the precipitate removed by centrifugation at 20,OOOg for 30 min. Solid ammonium sulfate, 6.5 gm, was slowly added to the resulting supernatant with continuous stirring at 0”. The suspension was stirred for 15 min at 0” and the precipitate collected by centrifugation at 20,OOOg for 30 min. The precipitate was dissolved in 10 ml of 0.01 M sodium phosphate buffer at pH 6.8 and 1 ml of 2% protamine sulfate was added dropwise with rapid stirring. The precipitate was removed by centrifugation at 20,OOOg for 20 min. The supernatant was combined with 15 ml of saturated ammonium sulfate, stirred for 15 min at 0”, and cleared of precipitate by centrifugation at 42,500g for 15 min. The phosphorylase was precipitated by the addition of 4 gm of solid ammonium sulfate, the solution was stirred for 10 min at O”, and the precipitate was collected by centrifugation at 42,500g for 30 min. The precipitate was dissolved in 5 ml of 0.01 M Tris-HCl, pH 7.5, and dialyzed for 8 hr against 6 liters of the same buffer at 0”. The dialysis buffer was changed and the dialysis continued for another 12 hr. A small precipitate was removed by centrifugation. A 2.5 X 90 cm column of Sephadex G-200 was equilibrated with 0.1 M Tris-HCl, pH 7.5 (10 ml/hr) . The protamine-treated preparation, 5.5 ml, was applied to the column and 5 ml fractions were collected. Assays of fractions showed that 70% of the enzyme appears in fractions 17-22 (see Fig. 1). Those fractions were concentrated with a colloidion membrane filter (Sartorius, Brinkmann Instruments, Westbury, N. Y.) under vacuum to a volume of 9 ml. Assay (spectrophotometric) and protein determination showed a specific activity of 980 U/ml or a 4.6-fold en-
r
1
FIG. 1. Chromatography of sucrose phosphorylase on Sephadex G-200. Closed these points correspond to protein concircles represent data obtained for A,,,; centration. Open circles are plotted for data obtained using spectrophotometric assay for fructose released. Area containing sucrose phosphorylase is shaded. (See ‘LProcedures” for conditions.)
SYNTHESIS
OF
SPECIFICALLY
TABLE of Sucrose
Purification
A mnm A mnm Extract (15 gm)” Protamine SO& Sephadex G-200 (frac. 17-22)
0.58 1.40 1.72
LABELED
Volume
1 Phosphorylase
(ml)
Proteinb (mg/ml)
26 5.5 9
19.1 20.0 1.S
a See under “Procedures” for description b BY ANI. c Pi uptake assay. d Spectrophotometric assay.
235
SUCROSE
Units (ml)
Units (mg)
Units
1460’ 4220c 1770d
76.5 213 980
37,960 23,200 15,900
‘ib recovery 100 61 42
of each step and assay.
richment for the gel filtration step. This preparation was used for both the phosphorolysis of sucrose-U-14C and the synthesis of specifically labeled sucroses (see Table 1 for summary of purification j . Paper Chromatography All paper chromatography was done on Whatman 3MM paper, prewashed in 0.01 N HCl and ethanol. Chambers were pre-equilibrated with solvent 4 hr before use for descending chromatography. Fructose, glucose, G-l-P, sucrose, and phosphate were always included as internal standards. Mobilities are reported as: Rr = distance sample migrated/distance front migrated. R glucose= distance sample migrated/distance glucose migrated, or Rsuwose= distance sample migrated/distance sucrose migrated. The solvent systems employed were (volumetric parts) : I. II. III. IV. V. VI. VII.
Methanol/concentrated NH,OH (7/3). Ethyl acetate/pyridine/H,O (12/5/4). Phenol (H,O saturat’ed) . 1-Butanol/pyridine/H20 (6/4/3). Methanol/formic acid/H,0 (85/15/5). 1-Butanol/acetic acid/H,0 (5/2/3). Ethyl acetate/acetic acid/H,0 (3/l/3).
Preparative chromatograms were allowed to drip off the end of the papers for 4 to 6 hr. Values from analytical chromatograms are reported as R* since they were terminated when the solvent front approached 2-3 cm from the lower edge of the paper. Radioactive areas on chromatographs were located by scanning with a radiochromatogram scanner (model 7201, Packard Instrument Co., Downers Grove, Ill.) Phosphate was detected by the spray technique of
236
CHASSY
AND
KRICHEVSKY
Bandurski and Axelrod (11). Sugars were located on paper by spraying with orcinol spray (12) followed by the “phenaline” spray reported by Koch et al. (13). Radioactive products were eluted using spin thimbles (Terra-Marine Bioresearch Laboratories, La Jolla, Calif.) ; 4 to 5 water washes completely elute the radioactivity. Preparation
of G-l-P-14C
(U)
and Fructose-14C
(U)
The phosphorolysis mixture contained 5 ml 0.05 M potassium phosphate, pH 7.0, 2.38 pmoles sucrose-U-14C (1000 ,uCi, prepurified by chromatography in solvent II; NEN, Boston, Mass.), and 0.1 ml sucrose phosphorylase (177 units). The mixture was held at 30” for 20 min, then applied directly to the origin of 2 chromatography papers (15 X 50 cm) ; the chromatograms were developed with solvent I. The sugar-containing areas (Rf 0.85-0.95) and the G-l-P containing areas (Rf 0.42-0.68) were resolved from phosphate (Rf < 0.40) and minor (<2%) radioactive impurities (RI 0.11, O-0.03). Each area was eluted with H,O, concentrated under a N, stream, and streaked on chromatography paper. The sugar-containing paper was developed with solvent II. The sugars were visualized and the radioactivity located. The fructose area (fructose has RglucOSe 1.35, sucrose Rglucose 0.65) contained 1.8 pmoles (384 ,&i), representing 77% recovery based on the fructose content of t.he starting sucrose-U-14C. Test chromatograms in solvent III and solvent IV failed to reveal any contaminating sugars or other radioactive compounds. The G-l-P chromatograms were developed with solvent V. The radioactivity and phosphate and sugar detection techniques found a single component with an Rf of 0.43 corresponding to G-l-P (glucose = 0.59, fructose = 0.62, sucrose = 0.57, PO1 = 0.72). The yield after elution and concentration was 2.04 pmoles (428 PCi) or 86% based on the glucose content of the sucrose-U-14C. Test chromatograms developed with solvent VI and solvent I revealed no (<0.2%) radioactive impurities. Preparation
of Sucrose
Containing
W-Glucose
The 14C-G-1-P (2.04 pmoles, 428 &i) was dissolved in 0.95 ml 0.05 A1 Tris-HCl, pH 7.5, and 14.4 mg fructose (80 pmoles) was added. After the fructose dissolved, 0.05 ml enzyme was added and the mixture allowed to stand 20 min at 30”. The mixture was streaked directly onto two chromatography papers and developed with solvent II. The sucrose was located by carbohydrate and phosphate sprays and eluted with H,O. The yield was 1.49 pmoles sucrose labeled with 14C-glucose (313 pci), or 73% (based on G-l-P). The 14C-sucrose was reapplied to a single paper and developed with solvent VII. The product was freed from a trace of contaminating G-l-P and glucose (glucose has RBUCrOSB1.29,
SYNTHESIS
OF
SPECIFICALLY
LABELED
237
SUCROSE
fructose has RSUeFOBB 1.64, G-l-P has RSUCrOSBof 0.21). Localization, elution, and concentration returned 1.37 pmoles (287 ,&i) , or 67% (based on G-l-P). The product was radiochemically homogeneous (<0.2% impurities). Preparation
of Sucrose
Containing
W-Fructose
The fructose (1.83 pmoles, 384 &i) was dissolved in 1.0 ml 0.05 M Tris-HCl, pH 7.5. To the resulting solution was added 26.4 mg G-l-P (Na salt, sesquihydrate, 80 pmoles, Schwarz/Mann Biochemical, Orangeberg, N. Y .) . Sucrose phosphorylase (0.05 ml) was added and the solution allowed t.o stand 20 min at 30”. The product was applied directly to two papers and developed (solvent II). The sucrose labeled with 14C fructose was located, eluted, and concentrated to 0.5 ml (14.1 pmoles, 295 ,&i, 77%). This solution was streaked on chromatography paper and developed with solvent VII. The sucrose was localized, eluted, and concentrated to dryness (1.32 pmoles, 278 $Zi, 72%), it was radiochemically pure when checked with solvents II, III, and IV. The specifically labeled sucroses were stored at -4O”, dried on 3MM paper, or stored in 50% ethanol at -20”. Isotopic
Purity
of Labeled
Sucrose
Small samples of specifically labeled sucrose, containing 0.2 PCi, were hydrolyzed for 5 min at 100” in 1 N HCl. The acidic solutions were streaked on 5 cm wide strips of Whatmann 3MM paper and the chromatographs developed in solvent II. The solvent was allowed to drip off the end of the paper for 4 hr as done in preparative chromatography. Control (no enzyme) chromatograms wit.h 14C-glucose, l*C-fructose, and 14C-sucrose and mixtures of each were run simult.aneously. The glucoseand fructose-containing areas of each of the specifically labeled sucroses were cut from the chromatograph and placed directly in scintillation vials containing toluene-PPO counting solution (0.4y0 PPO). The distribution of radioactivity in the specifically labeled sucrose indicates TABLE
2
Glucose* Samplea Sucrose-U-% 14C-Glucose W-Fructose a Details b Denotes
labeled sucrose labeled sucrose
% total
cpm 1.15 2.34 1.79
Fractoseb
x
105
x
lo5
x 102
described under “Procedures.” area of chromatogram corresponding
52.8
cpm
y& total 47.3
>99.9
1.03 1.41
x x
105 102
>99.9
1.14
x
106
to mobility
of each sugar.
238
CHASSY
AND
KRICHEVSKY
more than 99.9% radioactive purity and specificity (Table 2). The same results were obtained using 5 IU invertase (30 min at 30”) instead of acid hydrolysis. RESULTS
AND
DISCUSSION
The growth and harvesting of L. mesenteroides is a straightforward operation. No special skills or equipment are necessary. The use of this organism, instead of P. s.ucchurophila, has several advantages. The initial extracts have 25 times as much enzyme and, therefore, about 25 times higher specific activity. The enzyme can be sufficiently purified in three simple steps. In addition, the purified preparations have very little hydrolytic activity; although it is known that water can replace phosphate in the breakdown of the glucosyl-enzyme complex (l-6), this was never significant with the conditions employed in this report. The method of breaking cells has been altered; a French Pressure cell gives 20% higher yield than grinding with alumina. The French Pressure cell can be replaced by any of several breakage methods if necessary (e.g., sonication, Braun homogenizer). The preparation of G-l-P directly from sucrose-UJ4C has two advantages. Only one enzyme need be used and the G-l-P thus prepared is much less expensive than the item of commerce. Fructose-UJ4C is very inexpensive but is salvaged here as a side product. The overall yields of usable labeled sucroses (72% and 67%) are quite high. The theoretical yield, based on a Keg of 0.05 for the P. saccharophila. enzyme, is about 75-80s. It may be that the slightly higher pH (7.5 versus 6.6) and the Tris buffer shift the equilibrium slightly. Paper chromatography gives very good recoveries under these conditions. Recently, the use of “spin thimbles” and 4 or 5 washes with water to elute the products improved recoveries. The intended use of the products required high isotopic purity. Hydrolysis and separation of the glucose and fructose moieties revealed (Table 2) that the labeling is quite specific. This represents an increased degree of confidence over equilibrium-exchange methods. Higher yields and complete certainty about the products might be obtained using sucrose synthetase (UDPglucose:D-fructose 2-glucosyltransferase, EC 2.4.1.13) but the enzyme (14) is more difficult to prepare and the cost of UDPG-W, necessary for the synthesis of (glucosyl-14C)-sucrose, is too high to be of practical value. SUMMARY
A simplified and improved method for the synthesis of G-l-P-14C and specifically labeled sucroses has been developed. Using a preparation of
SYNTHESIS
OF
SPECIFICALLY
LABELED
SUCROSE
239
enzyme easily purified from Leuconostoc mesenteroides, high yields o sucroses were obtained. The radiochemical purity was (61%-72%) f quite high. The specific conditions for bacterial growth, enzyme purification and sucrose synthesis (utilizing paper chromatographic techniques) are completely described. REFERENCES 1. HASSID, W. Z., M. DOUDOROFF, AND H. A. BARKER, J. Amer. Chem. Sot. 66, 1416 (1944). 2. DOUDOROFF, M., H. A. BAKER, AND W. Z. H~SSID, J. Viol. Chrm. 168, 725 (1947). 3. DOUDOROFF, M., J. Biol. Chem. 151, 351 (1943). 4. ABRAHAM, S., AND W. Z. HASSID, in “Methods in Enzymology,” Vol. IV (S. P. Colowick and N. 0. Kaplan, eds.), p. 502. Academic Press, New York, 1957. 4~. WOLOCHAW, II., E. W. PUTMAN, M. DOUDOROFF, W. Z. HASSID, AND H. A. BARKER, J. Biol. Chem. 180, 1237 (1949). 5. SILYERSTEIN, R.. J. VOET, D. REED, AND R. H. ABELES, J. Biol. Chem. 242, 1338 (1967). 6. Vom, J. A., AND R. H. ABELES, J. Biol. Chem. 245, 1020 (1970). 7. TANZER, J., B. CHASSY, AND M. KRICHEVSKY, Biochem. Biophys. Acta., 261, 379 (1972). 8. KNUDSON, D. J., AND C. F. SCHACHTELE. Bucteriol. Proc. 71, 126 (1971). 9. DOUDOROFF, M., in “Methods in Enzymology,” Vol. 1 (S. P. Colowick and N. 0. Kaplan, eds.), p. 225. Academic Press, New York, 1955. 10. FISKE, C. H., AND Y. SUBBAROW, J. Biol. Chem. 66, 375 (1925). 11. BANDURSKI, R. S., AND B. AXELROD, J. Biol. Chem. 193, 405 (1952). 12. BEVENUE, S., AND K. I. WILLIAMS. Arch. Biochem. Biophys., 34, 225 (1951). 13. KOCH. R. B.. W. F. GEDDES, AND F. SMITII, Cereal Chem. 28, 424 (1951). 14. CARDING. C. E., L. F. LELOIR. AND J. CHIRIBOGA, J. Biol. Chem. 214, 149 (1955).