THE JOURNAL OF BIOLOGICAL CI~EMI~TRY Vol. 250, No. 5, Issue of March 10, pp. 1833-1837, 1975

Printed in U.S.A.

Glycogen Synthase Can Use Glucose as an Acceptor*

(Received for publication, June 12, 1974)

E~UAR~O SALSAS~ AND JOSEPH LARNER

From the Department oj Pharmacology, UrGwsity of Virgiliia School of Medicine, Charlottesvzlle, Virginia 22903

SUMMARY

Glycogen synthase purified to homogeneity from rabbit skeletal muscle is essentially free of carbohydrate and shows no activity in the absence of added acceptor. It can use glucose as a substrate converting it to a glucose disaccharide in the presence of UDP-glucose as cosubstrate. The re- action is dependent on time, and on UDP-glucose, glucose, and enzyme concentrations. The product of the single step reaction co-chromatographs in two solvent systems with maltose. The glucose disaccharide produced in the reaction with UDP-[Wlglucose and nonradioactive glucose as well as with nonradioactive UDP-glucose and [W]glucose is labeled asymmetrically. The linkage is characterized as a-1,4 and therefore the disaccharide is identified as maltose.

Glycogcx~ sy11th:w (lJI)I’-glucose :glycogcn au-l,4-glucosyl- transfcnw, I*X 2 4.1 1 1) is rcgardetl as the cnzymc cwlusivc~ly resporisiblc for ckmgation of the oukr lx:~11~~11cs of glycogrri (1) While nu1ncrous enzymes c:~talyziiig the syntlic& of oligos;tc- charides from sugar 11ucleotitles have lxx11 tlcscrilwd (for :I revkw see It. Ikxlondcr, Rcfs. 2 d 3) the question of tlw de nouo synthesis of IKW molecules of glycogcn still remains open.

Although several unprimctl cr.1 ,4-glucan syntheses hnvc been rcportcd (4---g) l~~~:~usc of the difficulty in ruling out oligosac-

charidc contamination (for :L discussion see Ryrnan ant1 TVhclan

(10) and Huwker et al. (ll)), the chemical proof for de novo a-l ,4-glucan synthesis has not bec~i cstablishctl conclusively.

We report here the one-step syntlicsis of a glucose dis~tcclinritlc identified as maltose from UI ) 1’.glucose and glucose with a highly purified prep:lration of rabbit muscle glycogeri syntliase. This

would rcprcscnt tlic first chemically identified reaction of ui~-

primed a-l ,4-glucan biosyiithcsis.

EXPERIMESTAL PROCEUURES

Enzyme

Glycogen synthase I was prepared according to t,he method of Smith et al. (12). It cont,ains essentially no detectable carbohy- drate (less than I70 with respect to protein) and shows no act,ivity

* This work was supported partially by United States Public Health Service. National Institutes of Health. (irant AM-14334.

t A portion of this work was carried out while 15. S. was sup- ported-by a Fulbright grant awarded by the Comisi6n de Inter- cambio Cultural entre I’spafia y 10s Xstados Unidos.de AmBrica. P. Calve Sotelo, 20, Madrid-l, Spain.

in the absence of added acceptor. The minimum specific activity of the different preparations used was 10 units per mg of protein.

Enzymutic Assa!y using Glucose or Oligosaccharides as Acceptors

Since the ordinary filter paper assay for glycogen could not be \I&, a modification of the method of DeWulf et CLI. (13) was em- ploycd. The reaction mixture cont,ained UI)P-(14C]gIucose (25 to 200 IJRI), glucose (400 to 1,500 mu), Tris-IICI, 50 mM, pII 7.8, ISI>TA 5 mM, NazSOr 2 mu, and an appropriate amount of enzyme in a total volun~c of 300 ~1. At different, time periods, aliquots of 75 ~1 were withdrawn and mixed with 150 ~1 of 40 rnM acetic acid to stop the reaction. Approximately 500 mg of a mixture of ion ex- changers (I 1(45, OII-, and I l(120, II+, 10: 15, w/w) were added, the t,~tbcs stirred and left at room tcmpcraturc for 1 to 2 hours. To each tllbc 1 ml of IIzO was then added and the mixtures filtered through glass wool. From the filtrate 0.5- to 1.0.ml aliquots were introdrlced to scintillation vials containing 8 ml of Aquasol and count,ed in the Beckman E-230 scintillation count,cr.

Initial velocities were calculated from slopes of Iincar time courses (see Fig. 1 and “llesults”). Each time course was run in dllplicatc.

Isolation of I,abeletl Glucose Disaccharide

The gll~cosc disaccharide and maltose standards are separated from glr~cosc and other oligosaccharidcs by means of descending paper chrom;ttogr:lphy on Whatman No. 3MRI paper using buta- nol-pyridine-w:Ltcr ((i:G:G, v/v/v) as solvent (Solvent A) or ethyl acet:ttc-pyridinc-w:tter (120:50:40, v/v/v) as solvent (Solvent B). After 48 horlrs, a separation of approximately 3.5 cm between glu- cose and maltose and of approximately 2.5 cm between maltose and maltotriosc was achieved. The standards wcrc stained by means of alkaline silver nitrate (14). The rest of the paper was analyzed for radioactivity withollt, st,aining in a radiochromatogram scanner and the zones containing the radioactivity cut out. The papers were placed in glass scint,illation vials containing 10 ml of 0.5% 2,5-diphcnyloxazole (PPO) in toluene and the radioactivity counted in the Beckman LS-230scintillation counter.

Charactcrizution of Labeled Glucose Disaccharide

Asyrur,x?try of I,abeZing-The glucose disaccharide was reduced with sodium borohydridc (15). The labeled glucose disaccharide diluted with nonradioactive maltose (7.5 pmol) and the appro- priate standards (7.5 pmol) were treated with 820 pmol of NaBH4 in 1120 in a total volume of 1.7 ml and left overnight at 20”. Reac- tion mixtures were then adjusted to neutrality with 2 N HCI. Acetone (0.5 ml) was then added and the mixtures evaporated to dryness under reduced pressure. The residues were dissolved in 0.5 ml of 11~0, the pH again adjusted to neutrality, 1 ml of metha- nol added and re-evaporated to dryness under reduced pressure. Thislastprocedtlrcwas repeated five times. Thesampleswere then hydrolyzed to glucose and sorbitol with 1 ml of 2 N HCl at 100°C for 2 hours. After hydrolysis samples were lyophilized two times, neutralized with 1 N NaOH. and desalted bv nassage throueh a mixed bed ion exchanger (Amberlite MB-3, ?&IIincirodt). -The salt-free sugars were then lyophilized and dissolved in 90 ~1 of 1120. Descending chromatography was performed on Whatman No. 3MM papers using Solvent B for 24 to 30 hours. Chromato-

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grams were developed and radioactive areas located and counted as described above.

Nature of Glycosiclic Band-To find out which carbon atoms are involved in the glycosidic bond, the following Smith’s degrada- tion procedure was used (16). The glucose disaccharide pro- duced from UDP-[‘4C]glucose and glucose was isolated by means of two descending paper chromatographic runs using Solvent B. (The papers had been prewashed with water.)

The radioactivity associated with the maltose zone was ex- tracted with water. An aliquot was treated with 2.5 pm01 of NaI04 in a total volume of 300 ~1. After 60 hours in the dark at 4”, the sample was treated with KB3H4 for 2 hours in ice. After that, 50 NM of KBH4 (unlabeled) was added, and the mixture was incubated 2 hours in ice. At the end of the treatment, the pH was lowered to 1.0 with HCI and submitted to 100’ for 10 min. The samples were concentrated by evaporation under reduced pressure, deionized by high voltage electrophoresis, extracted five times with methanol, and chromatographed on paper using pyridine- ethyl acetate-water (160:576:184 v/v/v) as solvent (Solvent C). The isolated products were chromatographed with isoamyl ace- tate-acetic acid-water (3:3:1, v/v/v) as solvent (Solvent D). As a standard, unlabeled maltose (250 nmol) was treated simi- larly.

To ascertain the stereochemistry of the glycosidic bond, the following enzymatic treatments were done. An aliquot of the original isolated sample was treated for 1 hour at 30” with crude a-glucosidase which contains no p-glucosidase (Takamine Di- azyme, partially purified by Dr. W. A. Volk, University of Virginia School of Medicine. Another aliquot was treated with p-glucosi- dase (Worthington) for 1 hour at 30”.

Standards of maltose and cellobiose were treated as the un- knowns. The products were separated by descending paper chromatography using Solvent B.

RESULTS

Kinetics-The time course of the reaction (Fig. 1, Curve a) was linear for the 15-min time period studied. The straight line does not pass through the origin. This is accounted for by a high background of radioactive glucose as will be shown by product

0 5 10

Incubation time in minutes

15

FIG. 1. Time course of the enzymatic reaction. The reaction mixture contained UDP-114Clelucose 1 mM. elucose 300 mM. Tris- HCl50 mM, pH 7.8, EDTA 5n%, NazS04 2n& and the appropriate amount of glycogen synthase in a total volume of 300 pl at 30”. At the indicated times 75-J aliquots were withdrawn and treated as described under “Experimental Procedures”. For each time point an aliquot of the corresponding deionized mixture was counted with kquasol, Curve a (6---A), whereas another aliquot was lyophilized, dissolved in 50~1 of H20, and chromatographed as described under “Experimental Procedures” with Solvent A. The radioactivity at the zone corresponding to maltose was counted with 10 ml of 0.5% 2.5-dinhenvloxazole in toluene, Curve b (0-v). The recovery in the ch;omatography and the effi- ciency of the counting procedures were taken into account.

isolation. The reaction proceeds without any lag period. The velocity of the reaction was dependent on the amount of enzyme (Fig. 2), and on the concentrations of both UDP-glucose and glucose. When the velocities obtained by varying the concen- tration of UDP-glucose at a fixed concentration of glucose (1.2 M) were plotted in the double reciprocal manner, an apparent K, of 2 x 10m4 M for UDP-glucose was obtained (Fig. 3). When the velocities obtained by varying the concentrations of glucose at a fixed concentration of UDP-glucose (1 X 10e4 M) were plotted in the double reciprocal manner, an apparent K,,, of 9 X 10-i M for glucose was obtained (Fig. 4).

Product Isolation-In order to relate velocity increase directly to glucose disaccharide formation, aliquots of the same treated reaction mixtures which had been counted for radioactivity (Fig. 1, Curve a) were lyophilized, dissolved in 50 ~1 of HtO, and sub- jected to chromatography (Solvent A) as described above.

V cpm

min

I I I

0 4 10 20

Enzyme concentration manits/ ul

FIG. 2. Enzyme concentration dependence of the reaction. The reaction mixture contained UDP-[“Cl glucose 100 LIM. glucose 1.57 M, Tris-HC150 mM, pH 7.8, EDTA 5 m;, NatSO 2.mh;and 4, 10, or 20 milliunits of enzyme in a total volume of 300 ~1. The milliunits of enzyme were calculated using the standard test mix- ture (12). The velocity was calculated from the slope of a time course (3,6, and 9 min) run in duplicate for each enzyme concen- tration as described in Fig. 1, Curve a.

l/V ,010 r

5 10 20 40

“[UDP-glucose] mM

FIG. 3. Double reciprocal plot with UDP-glucose as variable substrate. The reaction mixtures contained glucose 1.2 M, Tris- HCI 50 mM, pH 7.8, EDTA 5 mM, Na2SOd 2 mM, and UDP-glucose in the following final concentrations: 25,50,100, and 200 PM. The velocity was calculated from the slope of a time course (3, 6, and 9 min) run in duplicate for each UDP-glucose concentration as described in Fig. 1, Curve a. The apparent K,,, for UDP-glucose calculated from the figure is 2 X lo-” M.

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l/V .003 -

403 -

I I I 0 .5 .l 2.5

“[Glucor.] M

FIG. 4. Double reciprocal plot with glucose as variable sub- strate. The mixtures contained UDP-glucose 100 pM, Tris-HCI 50 mM, pH 7.8, EDTA 5 mM, Na2SOd 2 mM, and the following glu- cose concentrations: 400, 780, 1180, and 1570 mM. The velocity was calculated from the slope of a time course (3,6, and 9 min) run in duplicate for each glucose concentration as described in Fig. 1, Curve a. The apparent K,,, for glucose calculated from the figure is 9 x 10-r M.

point it was considered necessary to completely rule out the possibility that the enzyme might use carbohydrate primer re- maining in the enzyme preparation (even though not detected) and synthesize a new chain which could be hydrolyzed to a disac- charide presumably by another enzyme of the amylase type. To examine this possibility the reaction was run with nonradio- active UDP-glucose and [i4C]glucose, since under these conditions no radioactivity would have been expected to be incorporated into the hypothetical carbohydrate primer and therefore none in the disaccharide either. For this experiment 1 mM UDP-glucose and 5 PCi of [i4C]glucose at a concentration of 75 pM were used. Prior to the experiment the [*4C]glucose was purified by paper chromatography using Solvent A. The rest of the conditions were as described above. The incubation time was 6 hours at 30”. The desalted reaction mixture was subjected to chroma- tography in Solvent A and as shown in Fig. 5b, in addition to the radioactivity present in glucose, a peak of radioactivity in the position corresponding to maltose was found. A control chromatogram was run with the same amount (5 FCi) of [14C]- glucose to exclude any possible contamination of the labeled glucose with labeled maltose.

cpm

0 io 2’0 3’0 40 cm

5;: 7

q , #j ,

0 10 20 30 40 cm

CE;;’ ‘G.

FIG. 5. Radioactivity distribution in the product of the reac- tion. The deionized mixtures containing the products of the reaction were submitted to paper chromatography as described under “Experimental Procedures.” a, chromatogram (Solvent A) of the product after reaction with UDP-[Wlglucose and glu- cose. Paper cut and counted with 10 ml of 0.5y0 2,5-diphenyl- oxazole in toluene. b, chromatogram (Solvent B) of the product after reaction with [Wlglucose and UDP-glucose. Paper cut and counted with 10 ml of 0.5% 2,LLdiphenyloxazole in toluene. In both cases the position of the stained standards is shown. G, glu- cose; M, maltose.

Radioactivity was found only in the positions co-chromatograph- ing with maltose and glucose (Fig. 5~). Similar results were ob- tained with Solvent 13. The radioactivity co-chromatographing with the glucose was within experimental error the same in all the samples. It is due to contamination of the UDP-[14Clglucose by [‘4C]glucose. In later experiments using a repurified UDP- [14C]glucose, no such contamination was observed.

When the radioactivity in the glucose disaccharide isolated by chromatography was plotted as a function of the original time of incubation a linearity was obtained (Fig. 1, Curve b) . As shown in Fig. 1, the velocity (Curse b) is 79% of that obtained prior to chromatography (Curve a). The recovery in the chromatog- raphy and the efficiency in the two counting systems (paper and solution) were taken into account.

Alternate Production of Labeled Glucose Disaccharide-At this

Asymmetry of Radioactive Label in Glucose Disaccharide-It could still be argued that [r4C]glucose might have been incorpo- rated into a contaminating primer carbohydrate by means of the reverse synthetic action of a glucosidase. In fact, the glycogen- debranching enzyme can incorporate glucose into glycogen (17). The chain could then be split hypothetically into disaccharide units by an enzyme of the amylase type.

The definitive proof for the direct biosynthesis of a glucose disaccharide from UDP-glucose and glucose lies in the demon- stration of the asymmetry of distribution of the radioactivity in the product.

When the radioactive glucose disaccharide (Ml) is produced from nonradioactive glucose and UDP-[14C]glucose the nonre- ducing glucose moiety should be labeled but the reducing glucose moiety should not. Conversely, when [14C]glucose and non- radioactive UDP-glucose are used, the glucose disaccharide (M2) should have the radioactivity only in the reducing glucose moiety, and the nonreducing moiety should not be radioactive.

Both kinds of glucose disaccharide (Ml and M2) as well as a symmetrically labeled maltose (M3) as a control were reduced with sodium borohydride and hydrolyzed to glucose and sorbitol as described above. After hydrolysis the nonreducing glucose moiety yields glucose and the reducing moiety yields sorbitol.

After chromatographic separation (Solvent U) the correspond- ing radioactive spots were cut and counted and the per cent of radioactivity in each is shown in Table I. Seventy-five per cent of the radioactivity in Ml was recovered in the glucose, whereas 83% of the radioactivity in M2 was recovered in the sorbitol. In the control maltose (M3) 48% of the radioactivity was recov- ered in the glucose and 52% in the sorbitol.

Characterization of Linkage-The definitive proof of the iden- tity of the glucose disaccharide with maltose lies in the identifica- tion of an a-l, 4 linkage between the two glucoses.

Accordingly, the products of metaperiodate oxidation and po- tassium borohydride (3H-labeled) reduction were identified by paper chromatography.

In the case of a 1,4 linkage glycerol is obtained from the non- reducing glucose, and erythritol from the reducing end (16). Both sugars have the 3H label, introduced in the reduction of an aldehyde group to alcohol. The aldehyde group is the result of the metaperiodate oxidation.

In the present experiment the glucose disaccharide used was

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TABLE I Asymmetry in distribution of radioactivity in enzymatically

biosynthesized glucose disaccharide

The glucose disaccharides produced from UDP-[Wlglucose and glucose (Ml) and from UDP-glucose and [Wlglucose (M2), as well as uniformly labeled maltose (M3), were reduced, hydrolyzed to their constituent monomers and chromatographed with Sol- vent B as described under “Experimental Procedures.” The only two spots, corresponding to glucose and sorbitol were counted in 10 ml of 0.5oj, 2,5-diphenyloxazole in toluene and the per cent in each one shown in the table.

Per cent radioactivity distribution

Ml M2 M3

Glucose Sorbitol

75 25 17 83 48 52

TABLE II

Radioactivity in erythritol and glycerol after Smith’s degradaliotb of glucose disaccharide

Two samples of glucose disaccharide obtained with UDP- [“Clglucose and glucose, and 250 nmol of maltose as standard were oxidized with metaperiodatc, reduced with potjassium [3H]borohydride, and hydrolyzed as &scribed under “Experi- mental Procedures.” The products were chromntographed using Solvent C, the papers were scanned for radioactivity, and the two radioactive spots, which coincided with the position of the eryth- ritol and glycerol standards, cluted and co\mted with Aquasol as scintillation solvent. The results are expressed as disintegra- tions per min (appropriate corrections were made for double labeling, efhciency, and quenching of the samples).

aH I

“C

Sample 1 dpm x 10-3

Erythritol. Glycerol

Sample 2 Erythritol. Glycerol.

Malt,ose standard Erythritol. Glycerol.

103.7 0.7 196.5 31.0

210.2 1.4 336.3 41.5

2257.6 2770.4

obtained from UI)P-[Wlglucose and glucose, therefore, is labeled in the nonreducing glucose, and the glycerol obtained should have i4C label in addition to the 3H label. The results of the experi- ment performed as described under “Experimental l’rocedures” are shown in Table II.

It can be clearly seen that all the r4C label is present in the glycerol, whereas the W label is present in the glycerol and erythritol. A control of unlabeled maltose similarly treated show the same distribution of 3H label.

The labeled glycerol and erythritol co-chromatograph with standards using Solvent C, as well as Solvent I>.

To show that the linkage is a rather than p, an aliquot was treated with a-. and P-glucosidases as described under “Experi- mental Procedures.” The results of the chromatography of the products are shown on Table III. JIaltose and cellobiose were used as controls of the glucosidase action. It can be seen that whereas a-glucosidase hydrolyzes the samples to glucose, p-glu- cosidase is without effect. Maltose was hydrolyzed to glucose

by the oc-glucosidase preparation whereas it remained mostly as maltose after treatment by the @glucosidase preparation. Ex- actly the opposite was found with cellobiose.

The results of these two experiments unequivocally prove the presence of an a-l,4 linkage between the two glucoses and there- fore the glucose disaccharide is identified as maltose.

DISCUSSION

Several unprimed a-l, 4-glucan syntheses have been reported in the literature. For instance, highly purified muscle phos- phorylase was reported to form (Y-1,4-linked glucose chains in the absence of any detectable primer (4). However, Abdullah et al. (18) have attributed this synthesis to trace contamination of the glucose l-phosphate with primer molecules.

Other reports were related to de nova synthesis using sugar nucleotides donors. Krisman (5) described the synthesis by an unpurified enzyme preparation from rat liver of a glucan protein using UDP-glucose as substrate in which no added primer was required. Preiss and coworkers (6, 7) reported the formation of polysaccharides from ADPglucose in the absence of added primer catalyzed by enzyme preparations from different plant sources under a specific set of conditions. Polysaccharide syn theses have been found in Aerobacter aerogenes capable of cata- lyzing de nova synthesis (8). A report of unprimed glucan bio- synthesis by a particulate enzyme from an Escherichia coli mu- tant has also recently appeared (9).

However, Schicfer (19) was able to show that the de nova starch synthesis by a starch synthctase from sweet corn was attributable to the presence of a primer associated with the en- zyme preparation. hi a recent paper Hawker ct al. (11) pointed out that most of the results suggesting de novo or unprimed syn- thesis could be explained by the presence of an endogenous primer.

In all these cases the authors look for a multistep cu-1,4-glucan synthesis. We restricted our work to a single step reaction. The results presented in this paper unequivocally prove the direct synthesis of a glucose disaccharide, identified as maltose from UDl’-glucose and glucose catalyzed by glycogcn-free glycogen synthase. Despite the fact that maltose is a much better sub- strate than glucose, its concentration remains negligible through- out the reaction period. This is probably the reason why no maltotriosc is found. It is interesting that in this system the K, for UDPglucose is of the same order of magnitude as when determined with glycogen as acceptor (12). In the experiment depicted in Table I a certain amount of cross-contamination is present. F$F:e think this is mainly due to an overlapping on the chromatogram as the RF values of glucose and sorbitol are rather similar in this solvent.

The experiment shown in Table III proves that the linkage has the LY stereochemical configuration. The presence of Wlabeled erythritol as product of the Smith’s degradation (Table II) un- equivocally proves the presence of a 1,4 linkage. Both findings make certain the presence of maltose as product of the enzymatic reaction described here.

Nevertheless, from the data in Table II there appears to be preferential labeling by tritium of glycerol produced both from samples and standards; however, it is more pronounced in the samples. One possible explanation is that glycerol was produced more easily than erythritol during the first part of the reduction, that was performed with labeled potassium borohydride. An- other possibility would be the concomitant formation of a disac- charide having an a-l,6 linkage (isomaltose) or an a-l,2 linkage. This would account for the production of more glycerol than

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TABLE III Treatment of samples and standards with a- and b-glucosidase The radioactivity in maltose and in glucose is shown for the

samples. The amount of staining (+, -, or traces) is shown for the standards. The two samples described in Table II, as well as maltose and cellobiose as standards, were treated with 01- and 0.glucosidases as described under “Experimental Procedures.” The products were chromatographed using Solvent B. The portions of the papers corresponding to the samples were scanned for radioactivity and the only two radioactivity spots, which coincided with the positions of maltose, cellobiose, and glucose standards, eluted and counted with Aquasol as scintillation solvent. The portions of the papers corresponding to the treated standards were stained with the alkaline silver nitrate reagent.

Sample 1

Sample 2

Maltose

Cellobiose

Maltose Glucose

CM

640 10,720 11,700 1,250

750 20,850 19,600 4,010

Slaining

- 4- + Traces + - - -t

erythritol, as 2 molecules of glycerol and none of erythritol would be produced from each molecule of this disaccharide. We favor this last explanation, namely, the production of some isomaltose, as we have found a shoulder to the main peak of maltose after the preparative chromatography which was included with the mal- tose zone when preparing the samples for chemical characteriza- tion. The mobility of this shoulder was in agreement with that of isomaltose. The meaning, if any, of this supposed isomaltose production remains to be established.

The very high concentrations of glucose necessary to achieve significant synthesis of maltose makes it unlikely that this reac- tion is effective in Go at least under the conditions that we think prevail in the cell. Nevertheless, it is important to realize that mechanistically it is possible for the enzyme to use acceptors as small as glucose, and then maltose, maltotriose, etc., successively (20, 21). The possibility of an as yet unrecognized activator or modifier of the enzyme that could conceivably bring the K, for glucose to within a physiological range must of course be consid- ered.

However, in distinction to the cases previously outlined where serious doubts have been raised because of possible contaminating

primer, in the present case the evidence strongly favors the direct biosynthesis of the disaccharide as already discussed.

Alternatively, the possibility must be considered that some other monosaccharide with a higher affinity for the enzyme may start the synthesis of the primer, with the provision that this portion is removed after the polysaccharide has grown sufficiently large in a manner reminiscent of DNA synthesis (22).

Acknowledgments-We wish to express our appreciation and gratitude to Dr. W. A. Volk of the Department of Microbiology, University of Virginia School of Medicine, for his helpful sugges- tions on the characterization of the maltose.

REFERENCES 1. LELOIR, L. F., OLAVAHRIA, J. RI., GOLDEMDERG, S. II., AND

CARMINATTI, H. (1959) Arch. Biochem. 81,508-520 2. DI~:DONDEH, 11. (1961) Annu. Rev. Biochem. 80,347-382 3. DEDONDI+ Ii. (1972) in Biochemistry of the Glycosidic Linkage,

(PIIUS, R., AND PONTIS, II., eds) pp. 21-78, Academic Press, New York

4. ILLINGWORTH, B., BROWN, I). II., AND CORI, C. F. (1961) Proc. Nat. Acad. Sci. U. S. A. 47, 469-485

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6. OZSUN, J. D., HAWKER, J. S., ,\ND PRI~:ISS, J. (1971) Biochem. Biophys. Res. Cowzmun. 43, 631-636

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9. CHAMBOST, J. P., FAVARD, A., AND CATTANISO, J. (1973) Bio- them. Biophys. Res. Commun. 66, 132-140

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11. H~WKEE, J. S., OZBUN, J. L., OZAKI, H., GHE:IZNLIISRO, IS., AND PRFXSS. J. (1974) Arch. Biochewb. Biovhus. 160. 530m5Fil

12. SMITH, C: H.; VIL~I-PALGI, C., Bm‘wi, N. Ei.i &CHLENDEII, K. K., ROSICNKIMNS, A. M., AND LAI~NER, J. (1972) Methods Enz ymol. 28, 530-539

13. D~WULF, H., STALMANS, W., AND HENS, II. G. (1970) Eur. J. Biochem. 16, 1-8

14. ROUYT, J., AND FRENCH, D. (1963) Arch. Biochem. Biophys. 100,451-467

15. SCHICK, M., AND LENNARZ, W. J. (1969) J. Biol. Chem. 244, 2777-2789

16. GOLDSTEIN, I. J., H,zu, G. W., LEXIS, B. A., AND SMITII, F. (1965) Methods Carbohyd. Chem. 6,361-370

17. LARNER, J., AND SCHLISELFELD, L. H. (1956) Biochim. Biophys. Acta 20, 53-61

18. AIIDULL~H, M., FISCHER, E. H., QUMEHI, M. Y., SLESSOR, K. N., AKD WHELAN, W. J. (1965) Biochem. J. 97,9P

19. SCHIEFER, S. (1973) Fed. Proc. 32,603 20. GOLDINU~E:RG, S. H. (1962) Biochim. Biophys. Acta 66,357-359 21. SALSAS, Ii:. (1974) Fed. Proc. 33,1238 22. WICKNER, W., BRUTLAG, D., SCHEKMAN, R., AND KORNBERG,

A. (1972) Proc. Nat. Acad. Sci. U.S. A. 69,965-969

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E Salsas and J LarnerGlycogen synthase can use glucose as an acceptor.

1975, 250:1833-1837.J. Biol. Chem. 

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