CONVERSION OF MONOSACCHARIDES TO SUCROSE AND CELLULOSE IN WHEAT SEEDLINGS*

BY J. EDELMAN,t V. GINSBURG, AND W. Z. HASSID

(From the Department of Plant Biochemistry, College of Agriculture, University of California, Berkeley, California)

(Received for publication, October 4, 1954)

The conversion of monosaccharides to sucrose in the dark has long been studied by introducing solutions of hexoses into leaves or fragments of plant organs. It has been established that sucrose can be synthesized in viva in plants at the expense of monosaccharides such as glucose, fructose, or mannose (1). This observation has been recently confirmed with ran- domly C!14-labeled monosaccharides (2-4). When radioactive glucose or fructose was infiltrated into Canna leaf tissue (2), sucrose appeared to be readily formed from either monosaccharide. Analysis of the glucose and fructose derived from the hydrolysis of the sucrose showed that both mono- saccharide moieties were labeled. These investigations did not reveal, however, whether the monosaccharide introduced into the plant is directly incorporated into the sucrose molecule or whether it is first degraded to shorter chain compounds. The present paper reports further work on this problem in which glucose and fructose labeled in known positions were used.

Glucose-l-Cl4 was recently used by Greathouse (5) and by Brown and Neish (6) to investigate the synthesis of cellulose in plants. The experi- ments described by these workers were performed under conditions of slow rate of substrate absorption which necessitated long periods of incubation. Greathouse isolated the cellulose from cotton bolls 90 days after injection of labeled glucose, while Brown and Neish isolated the polysaccharide from wheat plants 24 to 96 hours after injection.

In the present investigation actively metabolizing wheat seedlings, 3 days old, were used. Absorption of the substrate by the seedlings was rapid and its transformation to other carbohydrates occurred readily; hence short periods of incubation (order of minutes) were adequate. C14- labeled monosaccharide gave rise to labeled sugar phosphates, sucrose, and cellulose in the tissue. These compounds were separated, their mono-

* This work was supported in part by a research contract with the United States Atomic Energy Commission.

t Fellow of the Rockefeller Foundation, 1953-54. Present address, Research In- stitute of Plant Physiology, Imperial College, South Kensington, London, S. W. 7, England.

843

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844 CONVERSION OF MONOSACCHARIDES

saccharide constituents were degraded, and the distribution of Cl4 in the molecules was determined.

Materials and Methods

Radioactive Sugars-Uniformly C14-labeled glucose was prepared by Dr. E. W. Putman (7) of this laboratory. The sugar was chromatographically pure and had activity of about 30 PC. per mg.

Glucose-l-Cl4 was prepared by an adaptation (8) of the method of Isbell et aZ. (9). It was chromatographically pure and had activity of about 7.5 PC. per mg. Degradation of the sugar (see below) showed that all the activity resided in carbon atom 1; the activity in the rest of the molecule was less than 1 per cent of the total.

Glucose-&Cl4 was obtained from Dr. H. S. Isbell (National Bureau of Standards). Its activity was about 1 PC. per mg.

Fructose-l-C14 was prepared by enolization of glucose-l-Cl4 (8). The labeled sugars were diluted with anhydrous glucose or fructose

(analytical grade) when necessary. Estimation of Radioactivity-Samples were counted directly on Whatman

So. 1 filter paper, with a Tracerlab rate meter (SU-3A) supplied with a Geiger tube (2). This instrument can be read with an accuracy of f5 per cent of the full scale deflection in ranges of 200 to 20,000 c.p.m. More accurate estimations were made on barium carbonate samples or sugar films mounted on aluminum disks, 2.5 cm. in diameter, by means of stand- ard end window Geiger counting equipment. Unless otherwise stated radioactivity measurements are expressed as mean values of duplicate samples corrected for self absorption, daily counter variation, and back- ground.

sugar Analysis-Reducing sugar was estimated by the method of So- mogyi (10) as modified by Nelson (11). Fructose was determined either by the modified method of Roe (12) or by that of Roe et al. (13).

Preparation of Wheat Seedlings-Wheat grain, variety Big Club, which was previously stored in air at room temperature, was soaked in tap water overnight, spread on moist filter paper, and left in the dark. After 3 days, at which time the coleoptiles were about 2 cm. long and three roots were normally present, each seedling was removed from the grain and placed in tap water through which a rapid stream of air was passed. Aeration was continued for 2 to 4 hours, the water being frequently changed. Prelimi- nary experiments with randomly labeled glucose-C’4 indicated that seed- lings separated from the grain and used without the ensuing period of aeration showed an initial release of reducing mat,erial, followed by a lag in sugar uptake before the maximal absorption rate was reached. Seed- lings which had been separated and aerated absorbed sugar at a constant

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J. EDELMAN, V. GINSBURG, AND W. Z. HASSID 845

rate for several hours. The rate of absorption by seedlings attached to the grain was relatively low.

Dry weight was estimated on tissue dried in an air oven at 80” for 24 hours.

Incubation with CYLabeled Glucose-In preliminary experiments the seedlings were suspended in 1 per cent glucose solution; in later experi- ments 0.1 per cent solutions were used. The tissue suspension was vigor- ously aerated during incubation.

In a typical experiment, thirty seedlings (dry weight about 4 mg. per seedling) were plared in 4 ml. of the radioactive sugar solution in a vessel of 20 ml. capacity with a sintered glass bottom through which air was blown. After appropriate time intervals the seedlings were removed, washed in rapidly running tap water for at least 30 seconds, plunged into about 20 ml. of boiling 70 per cent ethanol, and maintained at boiling point for 5 minutes. The ethanol extract was decanted.

Extraction and Separation qf Ethanol-Soluble Components-After repeat- ing the extraction of the tissue with three further portions of 70 per cent ethanol, the extra&s were combined, filtered, and evaporated to a small volume on a steam bath and the concentrated solution was (about 5 per cent sugar) applied as an even band 17 inches long on a filter paper sheet (Whatman No. 1, 18 X 24 inches) parallel to the longer edge. The paper sheet was developed in butanol-acetic acid-water mixture (14) for 3 days and the radioactive material located by exposing the dried sheet to East- man Kodak “no screen” x-ray film. The appropriate bands were cut from the chromatogram and eluted with water and the products dried in vacua over calcium chloride.

Preparation of Samples for Degradation-Because of the substrate speci- ficity requirements of the organism used in the sugar degradation, it was necessary to convert all the labeled carbohydrate samples to free glucose.

Sucrose was first completely hydrolyzed to glucose and fructose with invertase (British Drug Houses invertase concentrate); the monosaccha- rides were separated chromatographically on paper sheets, located, and eluted as previously described. The fructose fraction was epimerized to glucose by heating (with or without fructose carrier) in a sealed tube in 0.3 ml. of 0.2 M sodium phosphate buffer, pH 7.5, at 100’ for 2 hours, after which time the resulting mixture was resolved chromatographically on paper. There is evidence that, no randomization of the hexose chain oc- curs during the cpimerieation process (IT,). The yield of glucose was about 10 p’er cent.

The hexose phosphat,es were resolved into two main bands on one-dimen- sional chromatograms with butanol-acetic acid-mater as solvent. The bands were eluted and hydrolyzed by incubation with phosphatase from

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846 CONVERSION OF MONOSACCHARIDES

General Biochemicals, Inc. (0.4 per cent) at pH 8.0 for 20 hours at 35” in the presence of toluene. One of the resulting solutions contained radio- active glucose and the other both glucose and fructose.

The residual tissue obtained after extracting the wheat seedlings with ethanol contained chiefly cellulose and hemicellulose. The tissue was freed from hemicellulose by stirring twice with 1 N sodium hydroxide for 24 hours. The residue was exhaustively washed with water, heated with 1 N sulfuric acid in a sealed tube for 6 hours at loo”, washed, and blotted between filter papers. Glucose was obtained by hydrolysis of this purified cellulose by adaptation of the procedure of Monier-Williams (16): The material was treated with 72 per cent sulfuric acid for 7 days at room temperature, and the resultant dark suspension was diluted to 0.7 per cent sulfuric acid, filtered, and refluxed for 15 hours. After cooling, the acid was titrated with barium hydroxide, with phenolphthalein as an indicator, and the barium sulfate precipitate was removed by filtration. The solu- tion was concentrated to a sirup in DUCUO, made up to suitable volume, and chromatographed. Glucose was the major radioactive constituent. The yield of chromatographically pure glucose was approximately 21 per cent, based on the tissue residue obtained after ethanol and alkali extrac- tion.

Degradation of Glucose-The method used for degradation of glucose was essentially that of Gunsalus and Gibbs (17). The active glucose was diluted with carrier to give a total of 0.5 mmole of sugar and dissolved in water (1 to 5 ml.) and about a 10 per cent portion was taken for measure- ment of total activity, which was obtained after complete oxidation to COZ by the persulfate method of Katz et al. (18). The remainder was subjected to heterolactic fermentation by Leuconostoc mesenteroides (17), which de- grades glucose as follows:

C8H1206 -+ CO2 + CHa.CI-IZOH + HOOC.CHOH~CHa (1) (2) (3) (4) (5) (6)

Incubation with the organism was carried out in sodium phosphate solu- tion at pH 6 at about 2 cm. pressure. Conditions were arranged so that the fermentation was complete in less than 1 hour. After incubation the cells were immediately removed by centrifugation.

The ethanol was obtained by distillation of the reaction mixture. Usu- ally it was sufficient to obtain the sum activity of carbon atoms 2 and 3 in which case the ethanol was oxidized directly with persulfate. When an estimate of the activity in each atom was required, the ethanol was oxi- dized in the presence of 100 mg. of potassium dichromate and 12 N sulfuric acid to acetic acid; the acetic acid was recovered by steam distillation and split to CO2 (C-3) and methylamine (C-2) with sodium azide and sulfuric

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J. EDELMBN, V. GINSBURG, AND W. Z. HASSID 847

acid (19). Methylamine was oxidized to CO2 with alkaline potassium permanganate (19).

The residual fermentation mixture after removal of the ethanol was con- centrated to about 1 ml. and taken up with 20 gm. of fused potassium hydrogen sulfate and the lactic acid eluted from a column of the resultant dry powder with ether. The ether was removed, and the lactic acid was oxidized with acid permanganate in aqueous solution (19) to yield CO2 (C-4), and acetic acid (C-5 and C-6) which was either directly oxidized by persulfate or further degraded as described above. CO2 was collected in standard sodium hydroxide and converted to barium carbonate and activity measurements were made on about 10 mg. samples mounted on aluminum disks (diameter, 2.5 cm.). Reagent blank values, which were rarely as high as 5 per cent for 0.5 mmole samples of glucose, were deter- mined at all stages.

Reliability of Method-With 0.5 mmole quantities of glucose the sums of activity of the individual carbon atoms determined by the microbiologi- cal method and the total activity obtained from persulfate oxidation agreed well. Degradation of uniformly CY4-labeled glucose by the bio- logical method gave values ranging from 15.2 to 16.7 per cent per carbon atom (theoretical, 16.7 per cent), with a total recovery of 97.2 per cent. Degradation of glucose-l-C I4 by the same method showed that all the activity resided in C-l (experimental value, 102 per cent). The sum of activity in the other 5 carbon atoms was about 1 per cent, the total recov- ery of activity being 103.1 per cent.

It was found that, when 0.1 mmole quantities were subjected to degrada- tion, recoveries were consistently lower (80 to 85 per cent), possibly owing to unavoidable contamination by atmospheric CO2 and other reagent blank values which become significant at this level.

Results

Distribution of Cl4 in Sucrose, Cellulose, and Hexose Phosphates-Analysis of wheat seedlings before incubation with sugar solutions revealed the following results: shoots, 1.6 per cent sucrose, 4.6 per cent fructose, and 2.4 per cent glucose; roots, 1.5 per cent sucrose, 1.9 per cent fructose, and 3.2 per cent glucose, calculated on the dry weight of the tissue.

Chromatographic analysis of the external medium, before and after in- cubation of the wheat seedlings with glucose-l-CY4, showed that glucose was the only active component; no other sugars could be detected. The ethanol extract of the tissue after incubation contained about 45 per cent of the dry weight of the seedlings. Paper chromatography showed that most of the activity of this extract resided in the sucrose fraction with some in the sugar phosphates and very little in the monosaccharides.

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848 CONVERSION OF MONOSACCHARIDES

In the experiment described in Table I resolution of the sucrose to its monosaccharide constituents showed that the glucose moiety (2.10 mg.) contained a total activity of 1.61 X lo6 c.p.m. (counted as sugar film), while the fructose moiety (2.24 mg.) contained a total of 1.44 X lo6 c.p.m.

The results of the distribution of Cl4 in the hexoses derived from sucrose and cellulose are presented in Table I. The data show that the percentage incorporation of Cl4 activity in C-l of the monosaccharide moieties of the sucrose and in the glucose of the cellulose was practically identical (78 per cent). The values for Cl4 activity in C-6 of the hexose units of these com- pounds were also approximately the same (15 per cent). Examination of the distribution of Cl4 between C-l and C-6 of the hexose units of sucrose,

TABLE I

Per Cent Distribution of Cl4 in Hexoses Derived from Complex Saccharides after Incubating Wheat Seedlings with Glucose-i-C14

50 seedlings were incubated for 30 minutes in 4 ml. of medium containing 3 mg. of glucose-l-C*4 with specific activity of 7.5 pc. per mg. The values were obtained by L. mesenteroides degradation and are expressed as percentages of total activity in BaC03 resulting from T: ,ersulfate oxidation of the sugar sample.

Carbon atom Glucose moiety of Fructose moiety of S”ClYXO SIxrOSe

1 78.0 2+3 1.9 4 0.5 5 0.0 6 15.6

Total recovery 96.0

77.8 1.7 2.5

15.3

97.3

Glucose from cellulose

78.0

3.3 0.0

14.5 .__

95.8

hexose phosphates, and cellulose as a function of time showed a decrease in activity of C-l with a corresponding increase in C-6 during the first 15 minutes. After that time the ratio of activity in the 2 carbons remained practically constant at least up to 2 hours (Table II).

Continuous incubation of wheat seedlings for 8 hours with uniformly C14-labeled glucose resulted in the distribution of Cl4 in glucose from cellu- lose shown in Table III. Glucose-l-Cl4 gave similar results to those shown in Table I, except that there was a rather greater randomization between C-l and C-6 during the longer period; the effect of incubation with glucose- G-Q4 was to randomize C-l and C-6 approximately to the same extent, but in inverse ratio.

Distribution of Cl4 in Glucose Moiety of Sucrose When Incubated with Fructose-I-C14-An experiment in which fructose-l-U4 was used as sub- strate (70 seedlings incubated for 2 hours in 10 ml. of solution containing

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J. EDELMAN, V. GINSBURG, AND W. Z. HASSID 849

8 mg. of fructose-l-C 14; total activity 6.5 PC.) gave similar results to those already described with glucose-1-CL4. Thus the glucose moiety of sucrose

TABLE II

Change in Distribution of Cl” in C-l and C-6 of Hexose with Time after Continuous Incubation of Wheat Seedlings with Glucose-l-C’4

50 seedlings were used for each of 8 and 15 minute samples; twenty for others. Incubation was carried out in 5 ml. of medium containing 3 mg. of glucose-l-W with specific activity of 7.5 PC. per mg.

Incubation time

min.

8

15 60

120

Cl{ ratio, &6 X 100

86 90 84 78 75 / 80 77 78 78 74 77 78

Fructose GlllCOSe from from hexose

S”UO% phosphates

C” ratio, &6 x ‘00

14 1 22 23

/ 10 22

i 22 26 i 23

-

GIUCOX ram hexose phosphates

16 20 22 22

TABLE III

Per Cent Distribution of C” in Glucose from Cellulose after Incubation of Wheat Seedlings with Uniformly CF4-Labeled Glucose, Glucose-l -Q4,

and Glucose-6-C14

In each experiment, forty-one seedlings were incubated for 8 hours in 4 ml. of medium containing 15 mg. of labeled glucose of total activity as follows: 15 PC. of uniformly Cr4-labeled glucose, 25 PC. of glucose-l-C4, 1 NC. of glucose-6-P, respec- tively.

Carbon atom Evenly labeled C”-glucose Glucose-l-C” Glucose-6-C”

1 16.0 70.9 17.1

2+3 34.6 4.0 9.1 4 17.1 2.2 0.8 5 18.3 0.2 0.0 6 17.5 22.3 72.2

Total recovery. 103.5 I

99.6 I

99.2

showed 71 per cent of the activity in C-l and 15 per cent in C-6. The rest of the carbon chain contained negligible activity; total recovery was 89 per cent.

Because of the limited Cl4 activity in the sucrose, enolization of the fruc- tose component to glucose was not practicable. The distribution of Cl4 in the fructose moiety was therefore not determined.

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850 CONVERSION OF MONOSACCHARIDES

Glucose - 6 -phosphate -Fructose -6 -phosphate Interconversion in Vitro- Wheat seedlings were grown in the usual manner, separated from the grain, and washed thoroughly with tap lvater. The tissue (25.5 gm. wet weight) was ground in a mortar and the homogenate was squeezed through muslin to yield 17 ml. of extract. The extract was centrifuged, dialyzed against four changes of tap water at 3’ for 45 hours, and stored frozen at -15”.

3.0

20 40 60 ” 120

Time in Minutes FIG. 1. Conversion of glucose-6-phosphate to fructose-6-phosphate by wheat

seedling extract. 0 represents appearance of fructose-6-phosphate when no NaF was added; A, when NaF was added. l represents appearance of inorganic phos- phate when no NAF was added; A, when NaF was added. The solutions con- tained, per ml., 13.5 pmoles of glucose-6-phosphate, 70 pmoles of triethanolamine- Versene buffer, pH 7.1, 0.125 ml. of dialyzed wheat seedling extract and, when pres- ent, 50 pmoles of NaF. Incubation carried out at 30”. Samples (0.5 ml.) delivered into 6 per cent trichloroacetic acid (2.0 ml.) at appropriate time intervals.

The concentration of protein in the extract was 2.6 mg. per ml. This extract was used to demonstrate the presence of hexosephosphate isomerase by the appearance of fructose when the extract was allowed to act on glucose-6-phosphate.

A solution containing glucose-6-phosphate, wheat seedling extract, and buffer, pH 7.1, was incubated at. 30”. Analysis of the extract showed that fructose appeared rapidly and reached a maximum after 30 minutes (Fig. l), at this time the proportions of ketose and aldose (calculated as the 6-phosphates) being 25.5 and 74.5 per cent, respectively. This ratio, which agrees with the known equilibrium in the presence of phosphohexose

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J. EDELMAN, V. GINSBURG, AND W. Z. HASSID 851

isomerase (20), remained constant during a further 90 minutes incubation. The initial velocity, calculated over the first 5 minutes, showed that 0.4 pmole of fructose-B-phosphate (about 12 per cent of the final concentration) appeared per minute per ml. The results were essentially the same in the presence or absence of 50 pmoles of NaF per ml. of solution. The libera- tion of inorganic phosphate was slow (0.4 pmole per ml. in the first 30 minutes), and this was considerably reduced (to 0.14 pmole per ml.) in the presence of NaF. There was no significant pH optimum in the range investigated, pH 6.5 to 8.0.

DISCUSSION

The results obtained with wheat seedlings are in agreement with those previously obtained in this laboratory with Canna leaf disks (a), showing that glucose and fructose taken up by the tissue do not enter the monosac- charide pool in the plant but may become phosphorylated at the time they enter the cells. The major proportion of the Cl4 taken up in the form of labeled monosaccharide appeared in sucrose and the sugar phosphates; in contrast to the experiments with Canna leaf disks comparatively little ac- tivity was incorporated into other ethanol-extractable compounds. A small proportion of the activity after feeding glucose-l-Cl4 was recovered in glucose separated from the hydrolysis products of cellulose.

In the experiments with wheat seedlings and glucose-1-CY4, degradation of the glucose and fructose moieties from sucrose and the glucose constitu- ent of cellulose and of hexose phosphates showed that the major proportion of activity resided in C-l, indicating that hexose residues may be directly incorporated into the complex saccharides. However, considerable ran- domization had occurred between carbon atoms 1 and 6 in the combined hexoses in the shortest time intervals (8 minutes for soluble sugars (Table II), 30 minutes for cellulose (Table I)). Little activity could be detected in carbon atoms 2 to 5 even after longer periods of incubation (2 hours for sucrose and hexose phosphates, 8 hours for cellulose). Thus the data show that approximately 9 to 15 per cent of the Cl4 label was incorporated into C-6 during the first 8 minutes, increasing to some 20 per cent during the following 7 minutes. There was little subsequent change in the ratio of labeling in C-l and C-6. Our results of randomization between C-l and C-6 in cellulose agree with those of Brown and Neish (6), but are in con- trast with those of Greathouse (5) who could find no randomization in glucose obtained from cellulose from cotton bolls for as long as 90 days after feeding glucose-1-C4.

The activity in C-6 may have been derived from the incorporation of a certain proportion of hexose which had been synthesized through randomi- zation of C-l and C-G via reversible functioning of part of the glycolytic

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852 CONVERSION OF MONOSACCHARIDES

pathway, involving recombination of the two triose phosphates. Although the per cent activity in C-6 may give a rough measure of this process, it is possible t’hat complete equilibration between the triose phosphates does not occur and consequently the proportion of the total combined hexose cycled through this pathway may be appreciably greater than is indicated. However, in view of the diminution of activity in C-l and the correspond- ing increase of activity in C-G during the initial time periods (Table II), it is unlikely that all the hexose units constituting the complex saccharides are derived via this pathway.

An alternative pathway for randomization of these 2 carbon atoms in the combined hexose may involve products derived from the monophos- phate shunt (21). In this scheme Cs fragments labeled at C-3, derived from glucose-l-G4 via glycolysis, would serve as acceptors of unlabeled CS fragments transferred from sedoheptulose by transaldolase, thus forming fructose+phosphate labeled at C-6. However, this mechanism could not give rise to labeling in C-l with glucose-6-C** as substrate; thus the finding that C-l and C-6 in the glucose of cellulose are randomized to the same extent (in inverse ratio) when glucose labeled at C-l or C-6 is given appears to preclude such a pathway.

It is interesting to note that in animals the administration of glucose- l-Cl4 gives rise to glycogen (22) or lactose (23) in which the hexoses are labeled almost exclusively in C-l, there being no indication of significant randomization between C-l and C-6 after several hours. With the same substrate Acetobacter xylinum forms cellulose in which the glucose is labeled in carbons 1, 3, and 4, with the major proportion located at position 1 (24).

Comparison of the labeling of the monosaccharide moieties of the sucrose formed from the glucose-l-U4 showed that the specific activity of the glu- cose was approximately 10 per cent higher than that of the fructose. In experiments with Canna leaf disks (2) the specific activity of the glucose moiety of the sucrose derived from the infiltrated radioactive glucose was approximately 50 per cent greater than that of the fructose, and, cor- respondingly, when labeled fructose was infiltrated, the reverse was ob- served. The smaller difference of Cl4 label in the two monosaccharide moieties of sucrose in wheat seedlings may be explained by the assumption that in this actively metabolizing tissue a more rapid equilibrium is estab- lished between the glucose and fructose precursors derived from the glu- cose-l-Cl*. This equilibration could be achieved by phosphohexose iso- merase activity which was demonstrated in the tissue extracts. From initial velocities derived under the experimental conditions used for meas- urements of this activity, it was calculated that extracts were able to produce an amount of fructose-6-phosphate equivalent to that of the

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fructose moiety of sucrose synthesized by a corresponding amount of tissue at a rate roughly 10 times faster than that of glucose uptake by the seedlings.

Since the distribution of activity among the individual carbon atoms in the glucose and fructose moieties of sucrose and the glucose constituent of cellulose is nearly identical, it is probable that the monosaccharide con- stituents of these higher saccharides share a common precursor.

Two mechanisms of sucrose formation are known at present to occur in vitro, viz. those of Pseudomonas saccharophila (25) and wheat germ (26). In both a glucose donor (glucose-l-phosphate or uridine diphosphate glucose, respectively) combines with free fructose to form sucrose with the elimination of phosphate or uridine diphosphate. As the free fructose in the plant never becomes significantly labeled, the sucrose would be expected to be labeled predominantly in the glucose moiety, should this type of mechanism be acting in wheat seedlings. Thus, our finding that both moieties of sucrose are similarly labeled would indicate that free hexose is not involved in its synthesis; nevertheless, the possibility of utilization of free radioactive monosaccharide formed in transient amounts at the site of sucrose synthesis cannot be entirely overlooked.

SUMMARY

The transformation of C14-labeled glucose and fructose was studied in actively metabolizing wheat seedlings during periods ranging from 8 minutes to 8 hours. The radioactive sugar was rapidly absorbed, giving rise to labeled hexose phosphates, sucrose, and cellulose. The sucrose was always approximately equally labeled in both moieties.

When seedlings absorbed glucose-l-C I4 C-l of the glucose and fructose , isolated from the sucrose and of the glucose from cellulose was found to contain the major proportion of activity (78 per cent after 30 minutes). C-6 contained virtually all the remainder (15 per cent), which may have been produced through breakdown of the hexose molecule to trioses and randomization of C-l and C-6. The intermediate carbons in the chains (C-2 to C-5) contained very little activity (less than 5 per cent); glucose from the hexose phosphate fraction gave comparable results. A similar distribution was found in the glucose moiety of sucrose after absorption of fructose-1-U4.

Randomization between C-l and C-6 in the glucose and fructose moieties of the sucrose and in the glucose constituent of the cellulose showed little change with time.

The presence of an active phosphohexose isomerase was demonstrated in wheat seedling extracts.

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BIBLIOGRAPHY

1. Hassid, W. Z., and Putman, E. W., Ann. Rev. Plant Physiol., 1, 109 (1950). 2. Putman, E. W., and Hassid, W. Z., J. Bzol. Chem., 207, 885 (1954). 3. Porter, H. K., in Williams, R. T., Biqlogical transformations of starch and cel-

lulose, Biochemical Society symposia, Cambridge, 11, 27 (1953). 4. Vittorio, I’. V., Krotkov, G., and Reed, G. B., Canad. J. Bot., 32, 369 (1954). 5. Greathouse, G. A., Science, 117, 553 (1953). 6. Brown, S. A., and Neish, A. C., Canad. J. Biochem. and Physiol., 32, 170 (1954). 7. Putman, E. W., and Hassid, W. Z., J. Biol. Chem., 196, 749 (1952). 8. Hers, H. G., Edelman, J., and Ginsburg, V., J. Am. Chem. Sot., 76, 5160 (1954). 9. Isbell, H. S., Kurabinos, J. V., Frush, H. L., Holt, N. B., Schwebel, A., and Gal-

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J. Edelman, V. Ginsburg and W. Z. HassidSEEDLINGS

AND CELLULOSE IN WHEATMONOSACCHARIDES TO SUCROSE

CONVERSION OF

1955, 213:843-854.J. Biol. Chem. 

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