THE JOURNAL OF Bro~cmcnr. CHEM~TRY Vol. 244, No. 6, Issue of March 25, PP. 1608-1616, 1969

P&ted in U.S.A.

Biosynthesis of a Cell Wall Glucomannan in Mung Bean Seedlings*

A. D. ELBEIN~ (Received for publication, September 26, 1968)

From the Department of Biology, Rice University, Houston, Texas 77001

SUMMARY

A particulate enzyme fraction was isolated from mung bean seedlings which catalyzed the transfer of mannose from guanosine diphosphate n-mannose-14C and glucose from GDP-D-glucose-14C into an alkali-insoluble glucomannan. The incorporation of 14C-glucose into the polysaccharide was dependent on the presence of GDP-D-mannose in the incuba- tion mixtures, whereas the incorporation of W-mannose was strongly inhibited by adding GDP-D-glucose. Mg++ was required for the incorporation of mannose but not for glucose incorporation; the optimum concentration of Mg++ was about 1 x 10e2 M. Both reactions showed a sharp pH optimum at 7.5 in Tris buffer and were slightly inhibited by phosphate buffer.

Complete hydrolysis of the insoluble polysaccharide syn- thesized from GDP-D-mannose-14C released most of the radioactivity as 14C-mannose, although traces of activity were also present in glucose. On the other hand, in the polysac- charide synthesized from GDP-D-glucose-14C and GDP-D- mannose, all of the radioactivity was in glucose. The in- soluble polysaccharide was characterized by the examination of the soluble 14C-oligosaccharides released by a variety of treatments including enzymatic hydrolysis, partial acid hydrolysis, or acetolysis. From the 14C-mannose-labeled polymer a number of oligosaccharides were isolated which had varying ratios of mannose to glucose from 1: 1 to 3 or 4: 1. A W-disaccharide was isolated and partially identified as P-D-mannopyranosyl-n-glucopyranose. An identical 14c-

disaccharide was isolated from the polymer synthesized in the presence of GDP-D-glucoseJ4C and GDP-n-mannose, except that the radioactivity was in glucose. The identity of these disaccharides indicated that 14C-mannose and W-glucose were incorporated into the same polymer. The disaccharides as well as the polymer were susceptible to hydrolysis by enzymes that cleaved 0 linkages. Methyla- tion and then hydrolysis of several of the 14C-marmose-labeled oligosaccharides or the W.!-mannose-labeled insoluble polymer liberated two radioactive methylated sugars which were tentatively identified as 2,3,4,6-tetramethyhnannose

* This work was supported by Grant (C-165) from the Robert A. Welch Foundation.

1 Public Health Service Research Career Development Awardee of the National Institute of Allergy and Infectious Diseases.

and 2,3,6-trimethylmannose, suggesting a 1 -+ 4-linked polymer. The linkage was confirmed by periodate oxidation studies. Complete oxidation of several of the 14C-mannose- labeled oligosaccharides followed by reduction and hydrolysis led to the formation of 14C-glycerol and *4C-erythritol, indicat- ing that the r4C-mannose was attached by 1 -+ 4 linkages. Radioactive erythritol was also obtained from a 14C-glucose- labeled oligosaccharide. Based on the mannose to glucose ratios in the larger oligosaccharides, the product formed from GDP-D-mannoseJ4C or GDP-D-glucoseJ4C (in the presence of GDP-D-mannose) appears to be a j%(l --f 4)-linked glucomannan containing 3 or 4 mannose units per glucose molecule.

Glucomannans have been shown to be cell wall components in a variety of plants, being found mostly in woods and seeds. They are part of the hemicelluloses and thus are considered to be in close association with cellulose in the plant cell wall (1). These polysaccharides consist mostly of /3-n-mannose and @-n-glucose joined in chains by 1 ---f 4 linkages (2-7). However, small amounts of other sugars, particularly galactose, have been found, and there may be linkages other than 1 -+ 4. Thus, Mills and Time11 (8) isolated a trisaccharide containing equal amounts of glucose, mannose, and galactose from a glucomannan, and Meier (9) reported the isolation of 6-O-ar-n-galactopyranosyl- n-mannopyranose. These polymers also vary with regard to the proportion of mannose to glucose but the ratio is usually about 2 to 5 mannose units per glucose molecule.

Previously, a particulate enzyme fraction was isolated from mung bean (Phaseolus aureus) seedlings which catalyzed the transfer of glucose from GDP-n-glucose-14G into an alkali- insoluble polysaccharide which was characterized as celhrlose (10). The incorporation of glucose into the alkali-insoluble fraction was stimulated by the addition of GDP-n-mannose, but under these conditions the product formed was distinguish- able from cellulose by examination of the oligosaccharides released by partial acid hydrolysis (11). It was later shown that this particulate enzyme fraction catalyzed the incorporation of the mannosyl portion of GDP-n-mannoseJ4G into an alkali-

1608

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insoluble polymer which was partially characterized as a glucomannanl (12).

The present paper describes the properties of this enzyme system which is responsible for the synthesis of the glucomannan and shows that GDP-n-mannose-14C is the mannosyl donor and GDP-n-glucose-14C is the glucosyl donor. However, GDP-D- glucose-l*C appears to be utilized in this system only in the presence of GDP-n-mannose. However, the incorporation of radioactivity from GDP-n-mannose-r4C is inhibited by the addition of GDP-n-glucose. The product of both reactions is a glucomannan in which the radioactive sugars are attached by p-(1 --) 4) glycosidic linkages. Based on the analysis of the higher oligosaccharides the polymer may contain 3 to 4 mannose units per glucose molecule.

EXPERIMENTAL PROCEDURE

Mate&&-GDP-n-mannose-14C and GDP-n-glucose-14C were prepared by a modification (13) of the method of Roseman et al. (14). GDP-n-mannose-14C was also purchased from New England Nuclear. All other sugar nucleotides were prepared as described (13). The following were kindly donated as indicated: a /3-mannanase, free of cellulase and ,&mannosidase, and a partially purified P-mannosidase containing slight /3- glucosidase activity by Dr. Elwyn Reese, United States Quarter- master Corps., Natick, Massachusetts; a p-(1 --) 4)-linked glucomannan by Dr. T. E. Timell, Syracuse University; 4-0- (P-n-mannopyranosyl)-D-mannopyranoside and 4-o-(p-n-glu- copyranosyl)-n-mannopyranoside by Dr. C. Bishop, National Research Council; a p-(1 -+ 4)-linked mannosyl trisaccharide by Dr. R. Whistler, Purdue University; 2,4,6-trimethylman- nose by Dr. W. Lennarz, John Hopkins University School of Medicine. All other chemicals were obtained from commercial sources.

Analytical Methods-Hexose was determined by the anthrone method (15), reducing sugar by the procedure of Nelson (16), n-glucose with glucose oxidase (Worthington), and protein by the method of Sutherland et al. (17). Radioactivity on paper was located with a Packard radioactive paper scanner and was quantitatively determined with a Packard liquid scintillation spectrometer.

Periodate oxidations were performed by a modification of the method of Hay, Lewis, and Smith (18). Two to 5 pmoles (as mannose) of 14C-oligosaccharide were placed in 1 ml of 0.05 M

sodium metaperiodate and allowed to react at 5” for 72 to 96 hours. Samples were then reduced by the addition of 0.5 ml of 0.1 M NaBH4. After 30 min at room temperature, a second 0.5-ml aliquot of NaBH4 was added. After 30 min, the reaction was stopped by the addition of HCl to a final concentration of 2 N and samples were hydrolyzed by heating at 100” for 2 hours. The HCl was removed under reduced pressure and samples were passed through columns of Dowex 50-H+ to remove Na+. The borate was removed by repeated addition and evaporation of methanol. Finally, samples were treated with mixed bed ion exchange resin (equal parts of Dowex 50-H+ and Dowex I-COs=) and then chromatographed in Solvent 1.

Methylation of the soluble oligosaccharides was performed as described by Stewart, Mendershausen, and Ballou (19) and

1 Presented in part at the 51st Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, 1967.

methylation of insoluble polymers as described by Hakomori (20). Hydrolysis of the methylated derivatives was done in 2 N methanolic HCl (19). Reductions were performed with NaBH4 as previously described (21). Excess borohydride was destroyed by the addition of HCl and, where indicated, samples were hydrolyzed in 2 N HCl at 100” for 2 hours.

Chromatographic Methods-Descending paper chromatography was done on Whatman No. 1 or Whatman No. 3MM paper. The following solvent systems were used: I, butan-l-ol-pyridine- 0.1 N HCI (5:3:2); II, butan-1-ol-pyridine-water (6:4:3); III, propan-1-ol-ethyl acetate-water (7: 1:2) ; IV, ethyl acetate- concentrated acetic acid-water (3 : 3 : 1) ; V, methylethylketone- concentrated acetic acid-water (8: 1: 1). For isolation of the oligosaccharides, papers were usually developed for 48 to 72 hours. In some cases, papers were subjected to multiple developments; that is, after chromatography in the appropriate solvent, papers were removed, dried, and returned to the solvent for a second and possibly a third development. The mobility of the various oligosaccharides is presented relative to the mobility of cellobiose which was used as a standard reference compound. Methylated hexoses were run on paper impregnated with 0.02 M sodium borate or on thin layer plates in the following solvent systems: VI, butan-l-01-0.1 M sodium borate (20:5, upper phase) ; VII, butan-1-ol-ethanol-water (5: 1:4, upper phase); VIII, water-saturated methylethylketone. Sugars and alcohols were detected with the alkaline silver nitrate reagent (22) and methylated sugars with aniline acid phthalate (23).

Preparation of Enzyme-Mung beans (P. aureus) were germinated in the dark in a moist chamber (11). The particu- late enzyme was prepared by grinding with sand, and the particles that sedimented between 4,000 and 30,000 x 9 were used in these experiments. This enzyme preparation was stable for 1 or 2 days at 0” but slowly lost activity on prolonged storage. All attempts to stabilize the enzyme were unsuccessful.

Assay of Glucomannan Synthesis-A typical incubation mixture for the incorporation of mannose contained 0.1 bmole of GDP- n-mannose-14C (15,000 cpm), 2 pmoles of MgC12, 5 pmoles of Tris-HCl buffer (pH 7.5), and 0.1 ml of particulate enzyme (about 100 to 200 pg of protein) in a final volume of 0.2 ml. Tubes were incubated at 37” for 15 min unless otherwise specified and the reaction was terminated by adding 1 ml of water and placing tubes in a boiling water bath for 5 min. The precipitate was isolated by centrifugation and extracted several times with 1 ml of hot 2% NaOH. After each extraction, tubes were cooled and the precipitate was reisolated by centrifugation. The alkali-insoluble precipitate was then washed several times with water and its radioactive content was determined.

For determining the incorporation of glucose from GDP-D- glucose-**C into the glucomannan, assay mixtures were as follows: GDP-n-glucose-14C, 0.02 pmole (15,000 cpm); GDP-D- mannose, 0.1 pmole; MgC12, 2 pmoles; Tris-HCl buffer (pH 7.5), 5 pmoles; and particulate enzyme, 0.1 ml, in a final volume of 0.2 ml. After incubation at 37” for 15 min, tubes were assayed as described above for mannose incorporation,

Preparation of 14C-Glucomannan-For the preparation of large amounts of 14C-glucomannan for characterization, incuba- tion mixtures were as follows: GDP-n-mannose-14C or GDP-D- glucose-14C, 10 pmoles (1.5 X lo6 cpm); MgClz, 100 pmoles; Tris buffer, 150 pmoles; and enzyme, 10 ml (from 100 g of bean sprouts), in a final volume of 12 ml. When GDP-n-glucose-14C was used as substrate, reaction mixtures also contained 30

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1610 Glucmannan Biosynthesis Vol. 244, No. 6

pmoles of GDP-n-mannose. After incubation for 60 min at 37”, the reaction was stopped by heating at 100” for 5 min and the glucomannan was isolated as described.

On occasion, the alkaline extraction was carried out with 200 ml of 10% KOH at room temperature for 24 hours rather than hot 2y0 NaOH. This procedure extracts the hemicelluloses which can then be further separated. Thus, hemicellulose A precipitates from the alkaline solution upon neutralization with acetic acid, while hemicellulose B is precipitated by the addition of 2 to 3 volumes of alcohol (24). The alkali-insoluble residue from this treatment, or from the 20/, NaOH extraction described above, contained the glucomannan as well as cellulose and it was not possible to separate these two polymers. However, the glucomannan was characterized by analysis of the oligosac- charides released as described below.

Preparation of 14C-01igosaccharides---The alkali-insoluble resi- due was placed in a dialysis bag with either a hemicellulase (Worthington) or a @nannanase preparation and the water- soluble, dialyzable oligosaccharides were isolated and pursed by paper chromatography first in Solvent III and then in Sol- vents I and II (7, 12).

Oligosaccharides were also obtained by acetolysis of the poly- saccharide as described by Wolfrom and Thompson (25). The insoluble residue (145 mg) was shaken for 48 hours in a mixture of concentrated acetic acid (17 ml), acetic anhydride (17 ml), and concentrated sulfuric acid (1.7 ml), At the end of this time, the mixture was poured into 100 ml of ice water and the acetylated sugars were extracted into chloroform. Sugars were deacetylated with 0.2 M sodium methoxide and the deacetylated sugars were isolated and purified as in the enzymatic hydrolysis.

I I I I I !5cl loo I50 200 250

PROTEIN CONCENTRATION (Jig) FIG. 1. Effect of protein concentration on the incorporation of

radioactivitv from GDP-n-mannose-14C. Incubation mixtures ., were prepared as described except that the amount of the par- ticulate enzyme fraction was varied. At the end of the incuba- tion, the alkali-insoluble polymer was isolated as described and its radioactive content was determined.

F

12 -

2 IO-

B

8

oz G -

GDPG (+GDPM)

MINUTES FIG. 2. The effect of time on the incorporation of radioactivity

from GDP-n-mannose-%, GDP-n-glucose-i4C, and GDP-D- glucose-% plus GDP-n-mannose into the alkali-insoluble poly- mer. Incubation mixtures were as described and at the times indicated tubes were removed and the alkali-insoluble material was isolated and assayed for radioactivity.

Finally, a series of oligosaccharides was obtained by partial acid hydrolysis with fuming HCl (10, 11).

RESULTS

Effect of Time and Protein Concentration on Glucomannan Synthesis-As shown in Fig. 1, the incorporation of mannose from GDP-n-mannose-i4G by the particulate enzyme system was proportional to protein concentration over about a lo-fold range. Although the incorporation of glucose from GDP-D- glucose-14C (in the presence of GDP-n-mannose) was also some- what proportional to protein concentration, at low levels of protein the amount of radioactivity incorporated was low and, therefore, results were not completely reproducible.

Fig. 2 compares the incorporation of radioactivity into the alkali-insoluble material when GDP-n-mannose-14C, GDP-D-

glucose-14C, or GDP-n-glucose-14C plus GDP-n-mannose was used as substrate. Radioactivity from GDP-n-mannose-14C was fairly rapidly incorporated during a 15-min period and then leveled off with time. While the product of this reaction has the same solubility properties as cellulose, it has been character- ized as a glucomannan. GDP-n-glucose-14C, on the other hand, when incubated alone with the particulate enzyme is utilized at a very low rate. The product of this reaction was previously shown to be cellulose (11). The addition of unlabeled GDP-D- mannose stimulated the incorporation of radioactivity from GDP-n-glucose-14C, but this increased incorporation was into the glucomannan rather than into cellulose. Although the incorporation of radioactivity from GDP-n-mannose-14C was fairly proportional to time, the incorporation of radioactivity from GDP-n-glucose-14C was not linear beyond a few minutes. Therefore, the kinetic data presented, particularly with respect

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to glucose incorporation, probably represent degrees of incorpora- tion rather than initial rates of incorporation. Thus, although the kinetic data with GDP-n-glucose-14C were completely reproducible, they may represent approximations of actual rates.

Requirements for Glucomannan Synthesis-GDP-n-mannose- 14C was the only active mannosyl donor of any of those tested. Thus, the following showed less than 5% of the activity ex- hibited by GDP-n-mannose-14C: TDP-n-mannose-14C, W-D-

mannose-6-P, W-n-mannose-l-P, or 14C-n-mannose. Likewise, GDP-n-glucose-14C could not be replaced by any other glucosyl donor. Thus, UDP-n-glucose-14C, TDP-n-glucose-14C, CDP-D- glucose-14C, or ADP-n-glucose-14C was inactive (less than 3% of the activity exhibited by GDP-n-glucoseJ4C) either in the presence or absence of GDP-n-mannose.

E$ect of Concentration of Various Substrates-The incorpora- tion of mannose was proportional to GDP-n-mannoseJ4C concentration to about 2 x 1V4 M and the K, for GDP-D- mannoseJ4C was estimated to be about 1 x 10m4 M (Fig. 3). Since the particulate enzyme incorporates GbP-n-mannose-14C into a variety of products in addition to the glucomannan, this K, value may be somewhat higher than the real one. The incorporation of radioactivity from GDP-n-mannose-14C was markedly inhibited by the addition of GDP-n-glucose, as shown in Fig. 4. Increasing concentrations of GDP-n-glucose caused an increased inhibition in GDP-n-mannose-14C utilization.

Fig. 5 shows the effect of adding GDP-o-mannose to incuba- tion mixtures containing varying concentrations of GDP-D- glucose-W. It can be seen that increasing amounts of GDP-D- mannose caused an increased incorporation of radioactivity from GDP-n-glucose-14C into the alkali-insoluble material. At higher concentrations of GDP-n-mannose, maximum incorpora- tion of radioactivity occurred when the ratio of GDP-n-mannose to GDP-n-glucoseJ4C was about 3:l. The Km for GDP-D-

/

/ Km=lxlO -4# 1

I / I I

I OO

I I I I I 2 4 6 6 IO

k FIG. 3. Effect of GDP-n-mannose-14C concentration on the

incorporation of radioactivity into the glucomannan. Conditions were as described in the standard assay except that the concen- tration of GDP-n-mannose-14C was varied. At the end of the incubation, the amount of radioactivity in the alkali-insoluble polymer was determined.

I ~xIO-~M GDPG

FIG. 4. Effect of added GDP-n-glucose on the incorporation of GDP-n-mannose-%. Conditions were as described in the standard assay except that the concentration of GDP-D-mannose- 1% was varied as indicated. In addition, incubation mixtures contained GDP-n-glucose as shown. At theend of the incubation, the alkali-insoluble material was isolated and its radioactivity was determined.

glucose-14C could be roughly estimated and it varied depending on the GDP-n-mannose concentration. Thus, at a GDP-D- mannose concentration of 2.8 X lo-” M, the Km was 7 x 10-e M

while, at a GDP-o-mannose concentration of 3 X low4 M, the K,,, was about 5 X lo+ M. Roughly as the concentration of GDP-n-mannose doubled, the K,,, for GDP-n-glucose-14C also doubled. However, it should also be noted from Fig. 5 that when the concentration of GDP-n-glucoseJ4C exceeded that of GDP-n-mannose an inhibition in the incorporation was observed, suggesting that a certain ratio of the two nucleotides is necessary for optimum activity.

A number of other sugar nucleotides were tested, as shown in Table I, for their ability to stimulate or inhibit the incorporation of GDP-n-mannose-14C and GDP-n-glucoseJ4C into the alkali- insoluble polymer. As already indicated, GDP-n-glucose completely inhibited the incorporation of GDP-n-mannose-14C when added in concentrations exceeding that of GDP-D- mannoseJ4C. This same effect was also observed when GDP-D- galactose was substituted for GDP-n-glucose. However, while GDP-n-mannose stimulated GDP-n-glucoseJ4C incorporation, GDP-n-galactose inhibited this incorporation and even reversed the effect of GDP-n-mannose. Other sugar nucleotides, such as UDP-n-glucose and to a lesser extent ADP-n-glucose, inhibited the incorporation of GDP-n-mannoseJ4C but stimulated the incorporation of GDP-n-glucose-14C (in the presence of GDP-D- mannose). The nature of these various interactions is not understood at the present time.

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1612 Glucomannan Biosynthesis Vol. 244, No. 6

\ ’ <X IO-% GDPM \

‘\

~&8X10-% GDPM

ov I I I I I I 0 2 8 8 IO

GDffi CkKENTFWlON (M x 105) 12

FIG. 5. Effect of GDP-n-mannose on the incorporation of radio- activity from GDP-n-glucoseJ%. Assays were as described except that varying concentrations of GDP-n-glucoseJ4C were used as indicated. These experiments were done at four different concentrations of GDP-n-mannose. After incubation, the alkali- insoluble polymer was isolated as described and its radioactive content was determined.

TABLE I E$ect of various nucleotides on polysaccharide synthesis

Incubation mixtures were as described and contained GDP-D- mannoseJ4C, GDP-n-glucose-W, or GDP-n-glucoseJ4C plus GDP-n-mannose. In addition, sugar nucleotides (0.2 pmole) shown at the left were added as indicated. At the end of the incubation, the alkali-insoluble polymer was isolated and its radioactive content was determined.

Addition

Activity in product from

GDPM-1°C’ GD;$;&$ + GDPG-WC I I

None. . . . . ....... 1700 GDPG.. ....... 30 GDPM.. ....... 537 GDP-Gal. ....... 30 UDPG.. ....... 203 ADPG.. ....... 900 CDPG.. ....... 1230 CDPM.. ....... 1900 TDPG.. ....... 1010 TDPM. ....... 1570 UDP-Gal. ....... 1326

cm 891

478 1202 1289 1211 1076 1315

885 879

o GDP-n-mannoseJ4C. 6 GDP-n-glucoseJ4C plus GDP-n-mannose. c GDP-n-glucose-14C.

200 4

791 83

120 220 189 222 194 188 224

8 I

6

$ OF I I I I 0 5 IO I5 20 I

t 0

Mg CONCENTRATION (Mxl$) FIG. 6. Effect of Mg++ concentration on the incorporation of

radioactivity from GDP-n-mannoseJ4C. Conditions were as described in the text except that the concentration of Mg++ was varied as indicated. After incubation the alkali-insoluble poly- mer was isolated and its radioactivity was determined.

PH

FIG. 7. Effect of pH on glucomannan synthesis. Incubation mixtures were as described in the text except that buffers (0.05 M final concentration) were as follows: 0, acetate; 0, phosphate; or X Tris. Shown here is the incorporation of radioactivity from GDP-n-mannose-14C into the alkali-insoluble polymer. The same curves were obtained when GDP-n-glucose-14C (plus GDP- n-mannose) was used as substrate except that less radioactivity was incorporated.

Requirement for MS*--Fig. 6 shows the effect of Mg++ concentration on the incorporation of GDP-n-mannose-14C. The reaction was strongly dependent on the addition of Mg++ with the optimum concentration being about 1 x 10V2 M. The incorporation of GDP-n-glucose-14C, either in the presence or absence of GDP-n-mannose, was not dependent on the addition of Mg*. This may indicate that there are two different transferases present in the particles: one catalyzing the transfer of mannose and another catalyzing the transfer of glucose.

Eflect of pH-Fig. 7 shows the effect of varying the pH on the incorporation of radioactivity from GDP-n-mannose-14C. The reaction showed a sharp pH optimum at 7.5 in Tris buffer and was slightly inhibited in phosphate buffer (10 to 15yo inhibition).

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Incubation Mixture TABLE II

(IApe Layer Chloroform Layer (1)

+ Residue

Residue

I

(GDPM t Polysaccharides)

110% KOH

-tract (3) Residue

I

(Hemicelluloses)

I Water Extraction 3-4 Times

I Insoluble Residue (4)

I Water Wash

(Cellulose + Glucomannan)

FIG. 8. Flow diagram showing the sequence of extractions used in the isolation of the alkali-insoluble glucomannan. The in-

corporation of radioactivity into these various fractions is shown in Table II.

The incorporation of radioactivity from GDP-D-glucose-14C in

the presence of GDP-D-mannose showed similar results with

respect to varying pH except that the amount of radioactivity

incorporated was much lower.

CHARACTERIZATION OF PRODUCT

In order to characterize the product synthesized from GDP-D- mannose-14C or GDP-n-glucoseJ4C plus GDP-n-mannose, large scale reactions were prepared with about 1.5 x lo6 cpm of substrate as described. Fig. 8 presents a flow sheet of the various extraction procedures used in the isolation of the alkali- insoluble material. During the course of these studies, it was observed that a significant amount of the radioactivity from GDP-n-mannose-14C was incorporated into a lipid fraction? Thus, after incubation with GDP-D-mannose-14C, the reaction

was stopped by heating at 100” for 5 min and then the whole reaction mixture was extracted overnight at room temperature

with about 10 volumes of chloroform-methanol (3: 1). The layers were separated by centrifugation and the chloroform layer was carefully removed. The nature of the radioactive lipids is now under investigation.* The insoluble material which remained at the interface, was suspended in the aqueous phase and the mixture was carefully warmed to remove any traces of chloroform. The insoluble material was then isolated by centrifugation. The water layer, which contained unreacted sugar nucleotides and their breakdown products as well as a high molecular weight radioactive material, was removed and the insoluble residue was suspended in 10% KOH and kept at room temperature overnight. This extraction procedure

2 R. Laine and A. D. Elbein, unpublished observations.

Distribution of radioactivity from GDP-o-munnose-W

FIkXtiOlP Total radioactivity

I

Chloroform-methanol (3:l)

Water extract Ethanol-insoluble. . .

Alkali extract (10% KOH)

Hemicellulose A.. Hemicellulose B.. .

Insoluble residue. . .

......

......

......

CM

22,000

100,000

70,000 30,000

120,000

0 Fraction numbers refer to flow sheet.

solubilizes proteins and hemicelluloses. The hemicelluloses also contained a significant portion of the radioactivity from GDP-D- mannose-14C, and the nature of these products is also under investigation. The insoluble material from the alkali extract was isolated by centrifugation and washed three to four times by suspending in about 25 ml of water and then isolating by centrifugation. The distribution of radioactivity from GDP-D- mannoseJ4C into these various fractions is presented in Table II. Similar results were obtained with GDP-n-glucoseJQ plus GDP-n-mannose except that less radioactivity was incorporated into each of these fractions.

The insoluble residue remaining after these extractions con- tained about 8% of the activity initially present in GDP-D- mannoseJ4C and about 0.5 to 1% of the activity from GDP-D- glucose-14C. In both cases, the radioactivity was insoluble in all reagents tested (water, dilute acid and alkali, organic sol- vents) with the exception of syrupy phosphoric acid and cuoxan. The radioactivity could be solubilized in these latter reagents

and was precipitated by the addition of water. Complete Hydrolysis of Polymer-Complete hydrolysis of the

insoluble residue in 6 N HzS04 at 100” for 2 hours released several sugars which were identified as glucose, galactose, and mannose by paper chromatography in three different solvent systems (Solvents I, II, and III). In addition, an unidentified sugar which migrated faster than mannose and similar to rhamnose in Solvent II was also observed. In one experiment, after hydroly- sis of the polymer and isolation of the sugars by paper chroma- tography, the ratio of glucose to mannose to galactose as de- termined by the anthrone procedure was 4: 2: 1. The mannose was further characterized as n-mannose by its reactivity with a D-mannose isomerase isolated from Mycobacterium smegmatis,3 and the glucose as D-ghCOSS by reaction with glucose ox&se. When GDP-D-mannose-‘4C was used as substrate, most of the radioactivity in the insoluble residue was found in mannose,

although traces of activity were consistently found in glucose. In addition, in this case, the unidentified faster moving sugar was also radioactive. When GDP-n-glucoseJ4C was the sub- strate all of the activity appeared to be in glucose, although on occasion very small amounts of activity were found in the

mannose area of the paper. In this case, no activity was found in the faster-moving sugar.

Isolation of YY-Oligosaccharides-Since the insoluble material

contained the 14C-polymer as well as cellulose, characterization was based on the analyses of 14C-oligosaccharides liberated by

* A. E. Hey-Ferguson and A. D. Elbein, unpublished observa- tions.

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such treatments as enzymatic hydrolysis, partial acid hydrolysis, and acetolysis. Fig. 9 shows a scan of the radioactive oligosac- charides obtained on paper chromatograms from the polymer synthesized in the presence of GDP-n-mannose-14C. Enzymatic hydrolysis liberated about 50% of the radioactivity in soluble form and this was found in a variety of oligosaccharides. The major radioactive peaks in this case were at the origin and in the mannose area, although two 14C-disaccharides (EM4, EM5) and a W-trisaccharide (EM3) were obtained in this way.

Partial acid hydrolysis led to solubilization of 30 to 50% of the activity, but the majority of this was in larger oligosac- charides (PMl, PM2). Thus, only small amounts of disac- charide and trisaccharide were obtained by this procedure.

Finally, the polymer was subjected to acetolysis and the oligosaccharides were then deacetylated. The major radioactive

01 0 IO 20 so

DISTANCE FROM ORIGIN (CM)

m amD C G M

FIG. 9. Radioactive tracing of the soluble oligosaccharides released from the polymer. The glucomannan, synthesized from GDP-n-mannose-%, was treated as follows: Tracing A, enzymatic hydrolysis; Tracing B, partial acid hydrolysis; Tracing C’, acetoly- sis. The soluble oligosaccharides were separated by paper chromatography on Whatman No. 3MM paper in Solvent III. The polymer synthesized from GDP-n-glucose-14C and GDP-D- mannose was also subjected to enzymatic hydrolysis and a some- what similar scan was obtained except that considerably less radioactivity was in the various oligosaccharides. Standard compounds shown unde? the line are: C, cellobiose; G, glucose; M, mannose.

TABLE III

Analysis of oligosaccharides isolated from polymer

I;&&ti;LX- W-Substrate R cellobiose, Solvent III

Em1 Em2 Em3 Em4 Em5 A4 PM1 PM2 EG4 PGl

-I I- GDP-mannose 0.0 GDP-mannose 0.21 GDP-mannose 0.89 GDP-mannose 1.10 GDP-mannose 1.27 GDP-mannose 1.08 GDP-mannose 0.00 GDP-mannose 0.18 GDP-glucose 1.09 GDP-glucose 0.0

I

-

1

_-

-

Method of hydrolysis

Enzymatic Enzymatic Enzymatic Enzymatic Enzymatic Acetolysis Acid Acid Enzymatic Acid

Molar ratid of mannose-glucose

2.86:1.00 3.75:1.00 1.71:1.00 1.04:1.00

No glucose 1.08:1.00

0.82:1.00

a Mobility relative to a cellobiose standard. b Ratios were calculated by the anthrone method after isolation

of the sugars by paper chromatography.

TABLE IV

Chromatography of disaccharides isolated from polymer

Synthesized from R celP in solvent

I II III __--

GDP-mannose-‘%. . . . . 1.40 1.54 1.33

GDP-glucose-X + GDP-mannose , . . . . 1.40 1.54 1.33

* Mobility relative to a cellobiose standard.

peak which accounted for 20 to 30% of the total activity was a disaccharide (A4). Smaller amounts of activity were found in some larger oligosaccharides.

Since the polymer synthesized from GDP-n-glucoseJ4C and GDP-n-mannose contained considerably less radioactivity, it was impossible to use all of the above procedures. However, by enzymatic hydrolysis it was possible to isolate a radioactive disaccharide (EG4) for partial characterization. In addition, partial acid hydrolysis liberated some larger r4C-oligosaccharides (PGl).

Each of the oligosaccharides obtained by these procedures was chromatographed first in Solvent I and then in Solvents II and III until homogeneous,

Analysis of 14C-OZigosacchuri&--Table III presents some data on the analysis of a number of the oligosaccharides isolated from the polymer as described above. The amount of mannose and glucose in each oligosaccharide was determined by the anthrone method after hydrolysis and separation by paper chroma- tography. In some of the larger oligosaccharides obtained by enzymatic hydrolysis (EMI, EM2), the ratio of mannose to glucose was about 3 or 4: 1, which may indicate that this is the ratio of these sugars in the polysaccharide. However, since it was not possible to separate the intact 14C-polymer from cellu- lose, a direct analysis was not possible and therefore the exact proportion of sugars could not be determined. It can also be seen from Table III that oligosaccharides EM4, EG4, and A4 all exhibited the same migration on paper chromatograms in Solvent III indicative of a disaccharide. In addition, each had a molar ratio of mannose to glucose of 1 :l. A second disac- charide, EM5, containing only mannose was also isolated from enzymatic hydrolysates but the amount of material was too small for further characterization. Finally, a trisaccharide, EM3, was obtained which had 2 mannose units for each glucose. In each of the oligosaccharides isolated from the polymer syn- thesized from GDP-n-mannoseJ4C, most of the radioactivity was in mannose, although traces of activity were found in glucose. On the other hand, in the oligosaccharides from the GDP-D- glucose-14C-synthesized polysaccharide, essentially all of the radioactivity was in glucose. Several of the oligosaccharides described in Table III were used for further characterization as described below.

Partial Characterization of DisaccharidesEvidence indicating that disaccharide EM4, synthesized from GDP-n-mannose-14C, and EG4, synthesized from GDP-n-glucose-14c plus GDP-D- mannose, are identical is shown in Table IV. Both compounds had identical rates of migration on paper chromatograms in three diierent solvent systems. Both EM4 and EG4, as well as A4, were hydrolyzed by a P-mannosidase but not by /3-glu- cosidase. Reduction of the disaccharides with NaBH4 followed

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Issue of March 25, 1969 A. D. Elbein 1615

by acid hydrolysis resulted in a loss in the ability to react with glucose oxidase, indicating that glucose was at the reducing end. This was further substantiated by the isolation of 14C-mannose from NaBH4 reduced EM4 and -4. Thus, the structure of these disaccharides appears to be fl-n-mannopyranosyl-n- glucopyranose. Since the amount of these compounds was limited, the glycosidic linkage could not be determined, but it is probably 1 --f 4 based on the data from larger oligosaccharides presented below.

Determination of Glycosidic LinkageSeveral of the l*C- mannose-labeled oligosaccharides (EMl, EM2, PMl) as well as the insoluble polymer were subjected to complete methylation along with an authentic p-(1 -+ 4)-linked glucomannan and yeast mannan. After methanolysis and then hydrolysis of the methyl glycosides, the methylated sugars were chromatographed in Solvent VI. In this solvent, good separation of classes of methylated hexoses was obtained; that is, dimethyl-, trimethyl-, and tetramethylhexoses had far different mobilities. Fig. 10 shows a scan of the radioactive products from PM1 and it can be seen that two major peaks were obtained which corresponded to a trimethyl and a tetramethylmannose. The 14C-trimethyl- mannose was rechromatographed on paper chromatograms and thin layer plates in Solvents VI and VIII and in all cases the mobility corresponded to that of the trimethylmannose obtained from the ,8-(1 + 4)-linked glucomannan. Similar results were obtained with EM1 and EM2. However, in all cases, it was difficult to separate completely the 2,3,6-trimethylmannose from 2,4,6-trimethylmannose or the methylated mannose from yeast mannan. Therefore, periodate oxidation was used to verify the glycosidic linkage.

Several of the higher oligosaccharides (EMl, EM2, PMl, PM2) were subjected to periodate oxidation followed by reduction and hydrolysis. The alcohols produced by this treatment were then separated by paper chromatography in Solvent I. Fig. 11 shows a scan of the paper chromatogram obtained from PMl. With each of the oligosaccharides, two radioactive peaks were observed which corresponded to erythritol and glycerol. Eryth- ritol could only arise from a 1 -+ 4-linked oligosaccharide, suggesting that the original polymer was a /3-(1 ---f 4)-linked glucomannan. In addition, the presence of radioactive erythritol and glycerol indicates that the enzyme system is adding more than 1 mannose unit per chain. In several cases, traces of

300 c n

li; ; ;I-i!, IO 20 30 40

DISTANCE FROM ORIGIN (CM)

GM mt@l@

YM aD 0m

FIG. 10. Radioactive tracing of the methylated sugars from the alkali-insoluble polymer synthesized from GDP-n-mannose-W. After methylation and hydrolysis, the radioactive products were chromatoeranhed in Solvent VI. Also shown are the methvlated

I -

sugars from a p-(1 + 4)linked glucomannan and yeast mannan. Similar tracings were obtained from several of the higher oligo- saccharides (EMl, EM2, PMl).

5 300

b t

I 00

I I I lo 20 30

DISTANCE FROM ORIGIN (CM)

FIG. 11. Radioactive tracing of the products obtained from PM1 by periodate oxidation. The sample was completely oxi- dized followed by NaBHd reduction and then hydrolysis. The alcohols were chromatographed in Solvent I and the paper was then scanned for radioactivity. Similar results were obtained from EMl, EM2, and PM2. Standards shown are: W, mannitol; M, mannose; E, erythritol; G, glycerol.

radioactivity remained in mannose, suggesting either the presence of periodate-stable 1 --f 3 linkages or possibly incomplete oxida- tion. The fact that the enzyme system adds at least 2 mannose units per chain was confirmed by the methylation studies.

Periodate oxidation was also performed on PGl obtained by partial acid hydrolysis of the polymer synthesized from GDP-D- glucose-14C and GDP-n-mannose. After reduction and hydroly- sis, the alcohols were isolated as described above. Radioactive peaks corresponding to erythritol and glycerol were again detected in addition to a smaller radioactive peak corresponding to glucose. The presence of glucose may indicate the presence of some periodate stable linkages (such as 1 + 3), or may indicate incomplete oxidation. However, the fact that 14C- erythritol was obtained indicates that much of the glucose is attached by 1 + 4 linkages. In addition, this experiment indicates that the enzyme system is adding more than 1 glucose unit per chain.

DISCUSSION

A particulate enzyme system from mung bean seedlings was shown to catalyze the transfer of mannose from GDP-n-mannose- 14C and glucose from GDP-n-glucose-14C into a /3-(1 + 4)-linked glucomannan which appears to be a cell wall component. Perila and Bishop (7) isolated a number of oligosaccharides by treat- ment of a p-(1 + 4)-linked glucomannan isolated from jack pine with a crude hemicellulase. The disaccharide obtained in greatest amount was 4-O-(P-n-glucopyranosyl)-n-mannopyrano- side, although smaller amounts of 4-O-f$-n-mannopyranosyl)- n-glucopyranoside and 4-O-@-n-mannopyranosyl)-n-mannopy- ranoside were also obtained. In the 14C-glucomannan synthesized by the mung bean enzyme system, enzymatic hydrolysis liberated two 14C-disaccharides which were partially characterized as /3-n-mannosyl-n-glucose and P-n-mannosyl-n- mannose. The absence of a /3-n-glucosyl-n-mannose disac- charide in the enzymatic hydrolysis of the 14C-glucomannan is probably not surprising since the only oligosaccharides that would be detected are those containing radioactivity. Although the glucomannans usually found in plants are soluble in alkali, the one synthesized by the mung bean system appears to be insoluble in alkali and, in fact, has the same solubility properties as cellulose. In fact, the possibility that mannose is covalently bonded to cellulose cannot be excluded by these experiments. It is interesting to note in this regard that Adams and Bishop

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1616 Glucomannan Biosynthes& Vol. 244, No. 6

(26) isolated small amounts of mannose and galactose as well as several pentoses from the a-celluloses of cotton, wheat, and hardwood pulp. These results have also been observed by others (27). Whether these other sugars are part of the cellu- lose molecule or exist in closely related polysaccharides is not known at the present time. In the alkali-insoluble material synthesized from mung bean seedlings, glucose, galactose, and mannose as well as a faster migrating unidentified sugar were detected. The location of the galactose or the unidentified sugar is not known since these sugars were not found in any oligosaccharides. Since GDP-n-galactose inhibited the in- corporation of both GDP-n-mannose-14C and GDP-n-glucose-14C, it is interesting to speculate that GDP-n-galactose may be the galactosyl donor in this system. The unidentified sugar appears to be synthesized from GDP-n-mannose-14C since it was radio- active from the polysaccharide synthesized from GDP-D- mannose-“C.

4.

5.

GYAW, M. O., AND TIMELL, T. E., Can. J. Chem., 38, 1957 (1960).

TIMELL, T. E., in R. L. WHISTLER (Editor), Methods in carbo- hydrate chemistry, Vol. 6, Academic Press, New York, 1965, p. 137.

6.

7. 8.

9. 10.

11.

12.

13.

ROGERS, H. J., AND PEREINS, H. R., Cell walls and membranes, E. and F. N. Spon, Ltd., London, 1968.

PERILA, O., AND BISHOP, C. T., Can. J. Chem., 39,815 (1961). MILLS, A. R., AND TIMELL, T. E., Can. J. Chem., 41, 1389

(1963). MEIER, H., Acta Chem. &and., 14, 749 (1960). ELBEIN, A. E., BARBER, G. A., AND HASSID, W. Z., J. Amer.

Chem. Sot., 89, 309 (1964). BARBER, G. A., ELBEIN, A. D., AND HASSID, W. Z., J. Biol.

Chem., 239, 4056 (1964). ELBEIN, A. D., AND HASSID, W. Z., Biochem. Biophys. Res.

Commun., 23, 311 (1966). ELBEIN, A. D., in V. GINSBURG AND E. NEUFELD (Editors),

Methods in enzymology, Vol. 8, Academic Press, New York, 1966, p. 142.

The incorporation of GDP-n-glucose-14C into the glucomannan is greatly stimulated by the addition of GDP-n-mannose. Stimulation in the utilization of one sugar nucleotide by another would be expected in the case of a mixed polymer such as has been shown for bacterial cell wall lipopolysaccharides (28-31). However, the inhibition of GDP-n-mannose-14C incorporation by GDP-n-glucose is unexpected but may be due to the fact that the enzyme system has a greater affinity for GDP-n-glucose than for GDP-n-mannose (based on K, values). Thus, the presence of GDP-n-glucose in high concentrations may tie up the enzyme and prevent GDP-n-mannose from reacting. In fact, when the concentration of GDP-n-glucose-14C approaches that of GDP-D- mannose, the incorporation of glucose itself is inhibited.

14.

:t 17:

18.

ROSEMAN, S., DISTLER, J. J., MOFFATT, J. G., AND KHORANA, H. G., J. Amer. Chem. Sot.. 83. 659 (1961).

GDP-n-mannose-14C has been shown to be the precursor for a cell wall mannan in Micrococcus lysodeilcticus (32) as well as for a cell wall mannan in yeast (33). In the former case, an isoprenoid compound containing mannose was shown to be an intermediate in this synthesis (34). In this regard, it is interesting to note that the particulate enzyme fraction from mung bean seedlings also catalyzes the incorporation of mannose from GDP-D- mannose-% into a chloroform-methanol soluble material.” Studies are in progress to elucidate the structure and function of this compound. GDP-n-mannose-*4C has also been shown to be utilized for the synthesis of various other lipids in bacteria. Lennarz and Talamo (35) have shown the synthesis of mannosyl diglycerides in M. lysodeikticus and Brennan and Ballou (36) have shown that extracts of Mycobacterium phlei utilize this nu- cleotide for the synthesis of mannophosphoinositides. Whether compounds of this type are formed in the mung bean is not known at the present time.

19.

20. 21.

22.

23. 24.

Lo~wus, F., Anal. Chem., 24,‘219 (1952). ’ NELSON, N., J. Biol. Chem., 163,375 (1944). SUTHERLAND, E. W., CORI, C. F., HAYNES, R., AND OLSEN,

N. S., J. Biol. Chem., 160, 825 (1949). HAY, G. W., LEWIS, B. A., AND SMITH, F. A., in R. L. WHISTLER

(Editor), Methods in carbohydrate chemistry, Vol. 6, Aca- demic Press, New York, 1965, p. 377.

STEWART, T. S., MENDERSHAUSEN, P. B., AND BALLOU, C. E., Biochemistry, 7, 1843 (1968).

HAKOMORI, S., J. Biochem. (Tokyo), 66, 205 (1964). ELBEIN, A. D., AND HEATH, E. C., J. Biol. Chem., 240, 1919

(1965) . TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S.,

Nature, 166, 444 (1950).

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PARTRIDGE, S. M., Nature, 164, 443 (1949). WHISTLER, R. L., AND FEATHER, M. S., in R. L. WHISTLER

(Editor), Methods in carbohydrate chemistry, Vol. 6, Aca- demic Press, New York, 1965, p. 144.

WOLFROM, M. L. AND THOMPSON, A., in R. L. WHISTLER (Editor), Methods in carbohydrate chemistry, Vol. S, Aca- demic Press, New York, 1963, p. 143. -’ ’

ADAMS, G. A.. AND BISHOP. C. T.. Tavvi. 38. 672 (19651. ALBER~HEIM, ‘P., in J. BONIER AND J: ‘E: VARNER‘ (Editors),

Plant biochemistry, Academic Press, New York, 1965, p. 151. WRIGHT, A., DANKERT, M., AND ROBBINS, P. W., Proc. Nat.

Acad. Sci. U. S. A., 64, 235 (1965). EDSTROM, R. D., AND HEATH, E. C., J. Biol. Chem., 242,358l

(1967). WEIMER, I. M., HIGUCHI, T., ROTHFIELD. L.. SALTMARSH-

ANDREW, M.,‘OSBORN, &I. J:, AND HORE~KE~, B. L., Proc. Nat. Acad. Sci U. S. A.. 64. 228 (1965).

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A. D. ElbeinBiosynthesis of a Cell Wall Glucomannan in Mung Bean Seedlings

1969, 244:1608-1616.J. Biol. Chem. 

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