- Email: [email protected]
[2-3H]Mannose Incorporation in Cultured Plant Cells: Investigation of L -Galactose Residues of the Primary Cell Wall
,. PlantPhysiol. Vol. 132. pp. 484-490(1988)
[2- 3H]Mannose Incorporation in Cultured Plant Cells:
Investigation of L-Galactose Residues of the Primary Cell Wall ELIAS
A.-H.
BAYDOUN
1
and STEPHEN C. FRY
Department of Botany, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, U.K. Received September 4, 1987 . Accepted October 10, 1987
Summary D-[2-3H]Mannose was tested as a precursor for the labelling of L-galactose residues in cell wall polysaccharides where D-galactose residues predominate. Cultured cells of spinach, rose and maize rapidly took up [2-3H]mannose (e.g. 50 % in 4 - 8 min). About 90 % of the 3H supplied was converted to 3H20, the remaining 10 % being incorporated into polymers. Of the 3H incorporated into spinach polymers, 43 % was recovered in D-mannose, 37 % in L-fucose and 5 % in L-galactose residues. D-Galactose residues were not labelled. The L-[3H]galactose residues appeared to be in the pyranose form, and were released from the polymer in the form of short oligosaccharides upon treatment with «Driselase». L-[3H]Galactose residues were found in the phenol/acetic acid/H2 0 soluble, cold water soluble, pectic and hemicellulosic fractions of the total cell polymers, and were thus not confined to anyone particular polymer. The D-galactose: L-galactose ratio in spinach polymers was ca. 70: 1.
Key words: Spinacia oleracea, Rosa sp. (<
© 1988 by Gustav Fischer Verlag, Stuttgart
curs exclusively as non-reducing termini; maize «fibre» hemicellulose contains the sequence L-Galp-(1-.4)-D-Xylp-(1 ....2)-LAraf(I-. ... ) linked to a xylan backbone (Whistler and Corbett, 1955). Nothing is known about the linkage of L-Gal in the primary cell wall. The biological role of L-Gal, if any, is completely unknown. However, an enzyme has been described which synthesises GDP-L-Gal from GDP-D-Man by epimerisation at C-3 and C-5 and this suggested that [3H]mannose could be a specific precursor with which to label L-Gal residues in vivo. Interest in the polymers of the primary cell wall has recently been fuelled by the discovery that certain domains of wall polysaccharides, when released in the form of diffusible oligosaccharides, possess biological activities (McNeil et al., 1984). The wish to investigate the natural occurrence (Fry, 1986), transport (Baydoun and Fry, 1985) and turnover (Bay-
L-Galactose in Primary Walls
doun and Fry, 1988) of such regulatory molecules, and to define their chemical structures, has prompted efforts to develop highly sensitive methods for their detection and characterisation. By far the most sensitive technique available is radioactive labelling of living cells followed by detection of wall components by scintillation-counting. Labelling can be either non·specific, where the precursor (e.g. D-[U-1 4C]glucose) is incorporated into all residues of the wall polymers, or specific, where the precursor is incorporated only into certain specified residues (Fry, 1988). Nonspecific labelling has the advantage that all organic wall components are made detectable, whether or not their presence was expected. Specific labelling has the advantage that fewer analytical steps are required to identify a particular residue; this can be valuable when the residues of interest are present in small quantities. Precursors for specific labelling include Lfucose [labels L-Fuc residues only (Roberts, 1968)], L-arabinose [labels L-Ara and D-Xyl residues (Neish, 1958; Fry and Northcote, 1983)] and uronic acids or myo-inositol [label DGalA, D-GlcA, D-Api, D-Xyl and L-Ara residues (Neish, 1958; see also Fry, 1985)]. Little use has been made of radioactive D-mannose as a precursor for wall polysaccharides. Roberts (1971) reported that in maize roots D-[U-14C]mannose was incorporated into DMan, L-Fuc and L-Gal, but not D-Glc, D-Gal, D-Xyl or L-Ara residues. However, in our preliminary experiments with cell cultures, [U-1 4C]mannose and [PH]mannose gave nonspecific labelling. [2-3H]Mannose has ben applied to the specific labelling of mannose residues in glycoproteins since the 3H is lost as 3H20 if the molecule takes the isomerase branch of the following pathway, but retained if it takes the branch leading to [3H]glycoprotein: D-[2- 3H]Mannose ~
D-[2- 3H]Mannose-6-P
Isomerase
D-Fructose-6-P (non-radioactive)
+ D-[2- 3H]Mannose-1-P
+
general cell metabolism
+
GDP-n-[2- 3H]Mannose
+ [3H]Glycoproteins
Materials and Methods Radioisotopes and cell cultures n-[2- 3H]Mannose (specific activity = 16 Ci/mmol) was from Amersham International pic, Amersham, Bucks. Its radiochemical purity was checked by PC in EAWand found to be 99.6 %. Cell suspension cultures of spinach (Spinacia oleracea L.) and rose (Rosa sp., cv. «Paul's Scarlet») were as used by Fry and Street (1980). Cell cultures of maize (Zea mays, Black Mexican sweetcorn) were maintained as described for rose. The [2-3H]mannose solution was supplied to the cultures, at 1-10 /LCi/ ml, without non-radioactive carrier (i.e., 0.6 - 6.0 /LM), for '" 4 h. For the collection of culture «filtrate», the cells were allowed to sediment under gravity for a few minutes; duplicate samples of the culture medium were mixed immediately with 10 volumes of the Triton scintillant, and two further samples were dried in vacuo, redissolved in the original volume of water, and then mixed with 10 volumes of Triton scintillant. Preparation ofpolymers To obtain total radioactive polymer, the cells were plunged into 4 volumes of absolute ethanol, solid D-mannose was added to 0.5 % (w/v), and the cells were washed with 80% (v/v) ethanol until the washings were no longer radioactive. The cells were finally washed with acetone and dried. Some polymer samples were fractionated as follows: 10 mg polymer was stirred with 5 ml AW at 25°C for 16 h, and the residue was washed with a further 2 x 3-ml portions of AW. To the pooled AW-extracts, 0.05 volumes of 10 % (w/v) ammonium formate and 5 volumes of acetone were added; the mixture was stored at 4 °C for 16 h. The precipitate which formed was washed in acetone and dried «
Hydrolysis
We report here that [2-3H]mannose is also useful for specific labelling of wall polysaccharides, the 3H being incorporated into D-Man, L-Fuc and L-Gal residues: GDP-D-[2-3H]Mannose
485
-----++
GDP-L-[3H]Fucose
For analysis of monosaccharide compOSition, polymers were usually hydrolysed in 2 M TF A at 120°C for 1 h. The TF A was removed in vacuo, and the residue analysed by Pc. For enzymic hydrolysis, 10 mg of [3H]polymer was shaken at 25°C for 16h in 1 ml of 1 % «Driselase» [a preparation from frpex lacteus containing numerous exo- and endo-glycosidases; (NH4)2S04-precipitated and de-salted (Fry, 1982)] at pH 4.5. Solubilised products ( > 95 % of the total 3H) were analysed by GPc.
+ GDP-L-[3H]Galactose
This enables a very sensitive and specific method to be applied to the study of L-Gal residues in the primary cell wall.
Galactose dehydrogenase To distinguish D- and L-[3H]galactose, the eH]galactose was purified by PC in BA W and EPW and incubated at 25°C for 16 h in 140/Ll of a solution containing 0.5 mM non-radioactive D-galactose, 2.0mM ,6-NAD+, 20mM Tris-acetate (pH 8.6), and 25 Units/ml D-
486
ELIAS A.-H. BAYDOUN and STEPHEN C. FRY
galactose dehydrogenase (from recombinant E. coli using the Pseudo· monas fluorescens gene; 1 Unit will oxidise Il'mol/min; Sigma Chemical Co. product no G-6637). The products were analysed by PC in EPW.
4
I"
E
'"'0
Chromatography PC was on Whatman no. 1, by the descending method in one of 5 solvents (BAW, BEW, EAW, EPW or ~W - see abbreviations). Peaks of 3H were located by scintillation-counting of strips (1 x 4 cm). Co-chromatography of 3H with internal markers was verified by recovery of the radioactive strips from the scintillant, washing in toluene, drying, and staining the non-radioactive marker. Monosaccharides were stained with aniline hydrogen phthalate, and aldonic acids with AgN0 3/NaOH (Fry, 1988). GPC was on a 45 x 1.5 cm column of Bio-Gel P-2, eluted with acetic acid/pyridine/water (1: 1: 23, v/v/v), pH 4.5. Internal markers of Blue Dextran and D-mannose were used.
Assay 0/ radioactivity Scintillants were 0.5 % PPO/O.OS % POPOP in toluene (for PC strips) and 0.33 % PPO/0.033 % POPOP in toluene/Triton XI00 (2: 1, v/v) (for aqueous samples). [3H]Polymers were hydrolysed in 2M TFA prior to assay for radioactivity.
x
3
2
E
Co <..l
J:
C')
Time(min)
Fig. 2: Uptake and conversion to 3H2 0 of [2- 3H]mannose (2.6I'Ci/ ml, added at time 0) by spinach cells growing with 2 % glycerol [circles] or 2 % glucose [squares] as sole carbon source. Total 3H (open symbols) and non-volatile 3H (solid symbols) in the culture filtrate was measured.
Results
Uptake and fate of[2_3H]Mannose [2-3H]Mannose (0.6 t-tM) rapidly started to disappear from the medium of glycerol-grown spinach cells (Fig. 1). After a short time (10-20 min), 3H started to reappear in the medium. This was shown, from its disappearance on drying, to be largely 3H20. The [2-3H]mannose had been 50 % taken up and/or processed to 3H2 0 within 4-8 min (Fig. 1). Conversion of [2-3H]mannose to 3H20 was shown to be a cellular process by the fact that prolonged incubation of [2-3H]mannose in freshly-collected culture-filtrate did not produce any 3H2 0. This confirmed that mannose was indeed taken up and metabolised remarkably quickly. Spinach cells grown on sucrose, glucose or glycerol showed similar results, but uptake was slower in the (slowergrowing) glycerol cells (Fig. 2). This implies that D-mannose was taken up and phosphorylated by a system that is not swamped by ca. 20 - 56 mM D-glucose, the 2-epimer of Dmannose. This is surprising in view of the fact that D-glucose will protect cells from the toxic effects of excess D-mannose (Goldsworthy and Street, 1965). Results similar to those shown in Fig. 2 were obtained with cultured cells of rose and matze. In addition to 3H20, some cellular [3H]polymer was produced (ca. 10% of the supplied [2-3H]mannose after 125 min). Ca. 1 % of the radioactive material reappearing in the culture medium was also shown by PC in EAW to be polymer (RF 0.00).
1.0
E
Co <..l
J: 0.5
C')
o~--------~---------+--------~ o 20 40 60 Time(min)
Fig. 1: Uptake of [2-3H]mannose (1I'Ci/ml, added at time 0) by spinach cells growing with 2 % glycerol as sole carbon source. The graph plots the amount of 3H recovered in the culture filtrate: 0 - - 0 = total 3H; e--e = non-volatile 3H; 1::,.--1::,. = 3H20 (by difference).
Monomer composition offH]polymers [3H]Polymer from glucose-grown spinach cells was dried, hydrolysed in 2M TFA, and analysed by PC in EPW. Five peaks of 3H were reproducibly found (Fig. 3) co-migrating with polymer, galactose, mannose, fucose and 2-O-methylfucose respectively. The putative galactose spot was confirm-
L-Galactose in Primary Walls Xyl
I-----<
GaiA
2.0
'E"
~
1.5
Gal
Gle
Ara
............ I-----I~........-......
Fue ~
487
Rha
I------t
Rib
1-----1
Man
D-Man
L-Fue
C')
'~
Fig. 3: Paper chromatography of acid-hydrolysate of total [3H]polymers from glucose-grown spinach cells fed [2-3H]mannose (10j.tCi/ml) for 4h. Solvent: EPW. The bars indicate the positions of authentic markers. Imm = chromatographically-immobile material.
1·0
)(
E
c.
.8. Imm :t 0·5
C')
Me-L-Fue?
0~-=~=-----~---=~----~~-=-----3~0~---------4~0==~~
ed as such by co-chromatography with authentic L-galactose in BAW, BEW and c/>W. To determine whether the [3H]galactose was D- or L-, a sample was purified by PC and incubated with D-galactose dehydrogenase in the presence of non-radioactive D-galactose and excess NAD+. PC of the products in EPW showed that the non-radioactive D-galactose had been completely converted to galactonic acid (RCal = 0.1- 0.2), but all the 3H still co-chromatographed with galactose, indicating that the pH]galactose was 100 % L-galactose. This agrees with the expected range of sugar interconversions described in the Introduction. The Man and Fuc were not analysed in detail, but are assumed to be D-Man and L-Fuc respectively (Roberts, 1971). The material with RFO.OO (Fig. 3) was re-hydrolysed (2M TFA at 120°C for 1 h; or 4 M TF A at 120°C for 2 h): this caused slight (5 - 8 %) hydrolysis to mannose, but the bulk of the material was essentially acid-resistant, and is unidentified.
Kinetics of labelling of the polymer components Spinach cells were incubated with [2-3H]mannose and samples of the pH]polymer were analysed at intervals. DMan and L-Gal residues started to be labelled with a lag of < 5 min (Fig. 4). D-Man residues ceased to accumulate further 3H after 1 h incubation. However, the L-Gal residues continued to accumulate 3H well after 1 h. The fact that L-Gal residues started to be labelled with a negligible lag, and linearly for ca. 20 min, suggests that the endogenous pool of non-radioactive GDP-L-galactose was rapidly turning over; we therefore cannot explain the continued labelling of L-Gal residues from 1- 3 h by slow turnover of the GDP-L-[2- 3H]galactose pool. An alternative explanation is that the D-Man -+ L-Gal conversion occurs after incorporation into polymer(s), as reported for the formation of L-guluronic acid residues by 5-epimerisation of polysaccharide-bound D-mannuronic acid ~esidues (Haug and Larsen, 1969). This requires further testmg. L-Fuc residues were labelled at a linear rate after a slightly longer lag (9 min), presumably reflecting the time taken to convert GDP-D-Man into GDP-L-Fuc. The unidentified ma-
em from origin
terial of RF 0.00 was labelled without a detectable lag, but then lost ca. 65 % of its 3H, indicating turnover of a portion of this material.
Graded acid hydrolysis of the L-Gal residues To discover whether the L-Gal residues were mainly in the pyranose or the more acid-labile furanose form, graded acidhydrolysis was performed. Aliquots of [3H]polymer were incubated at 120°C for 1 h at various pHs, and the products analysed by PC in EPW. L-Gal residues were released as monosaccharides at a rate intermediate between D-Man and L-Fuc (Fig. 5), both of which are known to occur as pyranosides. If the L-Gal residues are non-reducing termini, as suggested for other systems (Whistler and Corbett, 1955; Bretting et aI., 1985), our data show that L-Gal was pyranosidically linked. Furanosides would have been released by milder acid [see data for sucrose: Fig. 5].
Products ofDriselase digestion «Driselase» has proved valuable in the characterisation of sugar linkages in the plant cell wall (Fry, 1986, 1988). It converts all the major sugar components of the spinach cell wall to monosaccharides except for the a-Xyl residues of xyloglucan, which are released as the disaccharide xylosyl-a(1-+6)-glucose. Driselase contains little protease and esterase activity. Nothing was known about the susceptibility of LGal residues, or the polymers that bear them, to Driselase. Therefore, Driselase digestion products of [3H]polymer were fractionated by GPC on Bio-Gel P-2. Each fraction was then hydrolysed with 2 M TF A to reveal the [3H]monosaccharides present (Fig. 6). L-Gal residues were less effectively released than D-Man or L-Fuc, which were largely converted to monosaccharide. About 43 % of the L-Gal was recovered in the tri- to tetrasaccharide zone [d. elution-volume of maltotetraose (Fig. 6)], showing that Driselase was poor in L-galactosidase activity. Recovery of D-Man and L-Fuc in this zone was 9 and 7 % respectively. Only 10 % of the L-Gal was recovered in the totally-excluded (high-M r ) fractions, in contrast to D-Man and L-Fuc (31 and 20 % respectively). This in-
488
ELIAS
A.-H. BAYDOUN and STEPHEN C. FRY
Man
75 32 .
;.,
0
E 50 :J E
x
CIl
E
'0 25
'-
E
o~~----~----~--~
o
N
I
0
x
3
10
Fig. 5: Graded acid hydrolysis of eH]polymers obtained by feeding [2- 3H]mannose (lO/-lCi/ml) to glucose-grown spinach cells for 4h. Aliquots of the [3H]polymer were hydrolysed for 1 h at 120°C in various acid solutions; products were analysed as in Fig. 3. The yield of glucose + fructose from non-radioactive sucrose (Suer) is included for comparison. Imm = chromatographically-immobile material.
E
c.
0
J:
C')
2
Approx. pH
5
o~--
_____________________
10
Imm
L·Cal: D-Cal ratio in spinach polymers
5
o
2
distributed between all the other fractions (except a-cellulose), indicating that L-Gal residues are not confined to any one particular polymer.
3
Time (h)
Fig.4: Time course for incorporation of 3H from [2-3H]mannose (10/-lCi/ml) into four components of polymers by cells growing with 2 % glycerol as sole carbon source. The [2-3H]mannose was fed at time 0, and polymer samples were taken at intervals for analysis as shown in Fig. 3. Imm = chromatographically-immobile material.
Hydrolysis of non-radioactive spinach cells in TF A, followed by 2-dimensional PC in EPW and BAWand staining with aniline hydrogen phthalate, indicated a ratio of total galactose: fucose of ca. 10: 1. The data in Fig. 3 indicate a ratio of L-Gal: Fuc of 1 : 7. Therefore the ratio L-Gal: D-Gal was ca. 1: 70, or 1.4 % L-Gal. This value is considerably lower than that (12 %) reported for sycamore cells (Roberts and Harrer, 1973), but nevertheless confirms that a significant minority of the Gal residues are the L-enantiomer.
Discussion dicates that the «core» of the polymer that bears the L-Gal was highly susceptible to Driselase digestion and thus unlikely to be a protein.
Fractionation of L-galactose-containing polymers To obtain more information about the polymers that bear L-Gal residues, the [3H]polymers were fractionated. The partition of L-Gal residues between the fractions is compared with that of D-Man and L-Fuc (Table 1). As expected, much of the D-Man and L-Fuc was in glycoprotein. In contrast, much of the L-Gal was pectic; however, it was also broadly
D-Mannose is toxic at high concentrations, inhibiting growth and respiration and promoting starch synthesis, probably by sequestering much of the cell's phosphate supply as Man-6-P (Goldsworthy and Street, 1965; Chen-she et aI., 1975). This suggests that plant cells have little Man-6-P isomerase activity [see scheme in Introduction], a view supported by Roberts' (1971) observation that, in maize roots, D{U- 14C]mannose is not readily incorporated into derivatives of Fru-6-P [i.e. polysaccharide-bound D-Glc etc.]' In contrast, our results with cultured cells indicate that tracer levels of Man-6-P were isomerised very rapidly indeed: thus, D-[U-14C]mannose and D{PH]mannose were extensively
L-Galactose in Primary Walls
489
Table 1: Distribution of L-[3H]Gal, D-[3H]Man and L-eH]Fuc residues between various cellular polymer fractions. L-Gal D-Man L-Fuc kcpm*) % kcpm*) % kcpm*) % .pAW-soluble 21 47 141 43 18.7 565 Cold H 20 soluble 14.4 16 76 6 23 7 37.3 41 365 31 80 24 Pectin Hemicellulose 18.9 21 180 15 81 25 a-Cellulose 1.0 1.1 4 0.3 3 0.9
Fraction
30
Total (10 mg)
Man
*) kcpm
SO
E
100
1200
100
328
100
thousands of counts per minute
~ these three quantitatively minor residues of the wall polysaccharides.
til
-
=
90.3
20
0
Acknowledgements
;!1
Weare most grateful to Miss Sheenagh Bryson for excellent technical assistance. E. A.-H. B. thanks Yarmouk University, Irbid, Jordan for a research grant. S. C. F. thanks the EEC for the award of a research grant under their «Biotechnology Action Programme». 10
References
o~~
____________
0.25
~
__________
0.5 Column
volumes
~
___
0.75 eluted
Fig. 6: GPC on Bio-Gel P-2 of Driselase-digested [3H]polymers obtained by feeding [2- 3H]mannose (10 "Ci/ml) to glucose-grown spinach cells for 4 h. Each Bio-Gel fraction was acid-hydrolysed and analysed as in Fig. 3. Imm = chromatographically-immobile material. BD
I=
Internal marker Blue Dextran.
f--~M""---II
=
Internal marker D-mannose.
f----,M'-'.4-'---I1
=
Position of elution of maltotetraose (run under identical conditions but on a separate occasion) relative to Blue Dextran and D-mannose.
metabolised to derivatives of Fru-6-P, and 0-[2- 3H]mannose quickly lost ca. 90 % of its 3H as 3H2 0. Since we tested the [23H]mannose at low concentrations ( =:::: 6 I'M), our results do not say whether Man-6-P isomerase activity had a high V max. Nevertheless, the cultured cells were clearly able to mobilise the levels of Man-6-P formed from (non-toxic) tracer levels of exogenous mannose. Our results show that for carrier-free 0-[3H]mannose to be useful as a specific precursor of o-Man, L-Fuc and L-Gal residues, it must carry the label in the 2-position. Although only 10 % of the 3H is recruited into polymers, the rapidity of uptake and specificity of incorporation makes 0-[23H]mannose a valuable precursor for the investigation of
ANDERSON, E.: The preparation of I-galactose from flaxseed mucilage.]. BioI. Chern. 100, 249-253 (1933). BAYDOUN, E. A.-H. and S. C. FRY: The immobility of pectic substances in injured tomato leaves and its bearing on the identity of the wound hormone. Planta 165, 269-276 (1985). - - The in vivo turnover of xyloglucan nonasaccharide, a possible biologically active cell wall fragment. (In preparation) (1988). BRETTING, H., G. JACOBS, I. BENECKE, W. A. KONIG, and J. THIEM: The occurrence of L-galactose in snail galactans. Carbohydr. Res. 139,225-236 (1985). CHEN-SHE, S., D. H. LEWIS, and D. A. WALKER: Stimulation of photosynthetic starch formation by sequestration of cytoplasmic orthophosphate. New Phytol. 74, 383 -392 (1975). FRY, S. c.: Phenolic components of the primary cell wall. Feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharides. Biochem. J. 203, 493 - 504 (1982). - Primary cell wall metabolism. Oxford Surv. Plant Mol. Cell BioI. 2,1-42 (1985). - In-vivo formation of xyloglucan nonasaccharide: a possible biologically active cell-wall fragment. Planta 169, 443-453 (1986). - The Growing Plant Cell Wall: Chemical and Metabolic Analysis. 333 pp. Longmans, London (1988). FRY, S. C. and D. H. NORTHCOTE: Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides. Plant Physiol. 73, 1055-1061 (1983). FRY, S. C. and H. E. STREET: Gibberellin-sensitive suspension cultures. Plant Physiol. 65, 472-477 (1980). GOLDSWORTHY, A. and H. E. STREET: Carbohydrate nutrition of tomato roots. VIII. The mechanism of the inhibition by D-mannose of the respiration of excised roots. Ann. Bot., N. S. 29, 45-58 (1965). HAUG, A. and B. LARSEN: Biosynthesis of alginate. Epimerisation of D-mannuronic acid to L-guluronic acid residues in the polysaccharide chain. Biochim. Biophys. Acta 192,557 -559 (1969). McNEIL, M., A. G. DARVILL, S. C. FRY, and P. ALBERSHEIM: Structure and function of the primary cell walls of plants. Annu. Rev. Biochem. 53, 625-663 (1984).
490
EUAS A.-H. BAYDOUN and STEPHEN C. FRY
NEISH, A. c.: The biosynthesis of cell wall carbohydrates. IV. Further studies on cellulose and xylan in wheat. Can. J. Biochem. Physio1.36, 187 -193 (1958). ROBERTS, R. M.: The metabolism of L-fucose by sycamore (Acerpseudoplatanus L.) cell cultures. Arch. Biochem. Biophys. 128, 818-820 (1968). - The metabolism of D-mannose- 14C to polysaccharide in corn roots. Specific labeling of L-galactose, D-mannose, and L-fucose. Arch. Biochem. Biophys. 145, 685-692 (1971).
ROBERTS, R. M. and E. HARRER: Determination of L-galactose in polysaccharide material. Phytochemistry 12, 2679-2682 (1973). WHISTLER, R. L. and W. M. CORBETT: Oligosaccharides from partial acid hydrolysis of corn fiber hemicellulose. J. Am. Chern. Soc. 77,6328-6330 (1955). WINTERSTEIN, E.: Ueber die aus Chagual-Gummi entstehenden Glucosen (inactive Galaktose und Xylose). Ber. deut. Chern. Ges.31, 1571-1573 (1898).
[2- 3H]Mannose Incorporation in Cultured Plant Cells:
Investigation of L-Galactose Residues of the Primary Cell Wall ELIAS
A.-H.
BAYDOUN
1
and STEPHEN C. FRY
Department of Botany, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, U.K. Received September 4, 1987 . Accepted October 10, 1987
Summary D-[2-3H]Mannose was tested as a precursor for the labelling of L-galactose residues in cell wall polysaccharides where D-galactose residues predominate. Cultured cells of spinach, rose and maize rapidly took up [2-3H]mannose (e.g. 50 % in 4 - 8 min). About 90 % of the 3H supplied was converted to 3H20, the remaining 10 % being incorporated into polymers. Of the 3H incorporated into spinach polymers, 43 % was recovered in D-mannose, 37 % in L-fucose and 5 % in L-galactose residues. D-Galactose residues were not labelled. The L-[3H]galactose residues appeared to be in the pyranose form, and were released from the polymer in the form of short oligosaccharides upon treatment with «Driselase». L-[3H]Galactose residues were found in the phenol/acetic acid/H2 0 soluble, cold water soluble, pectic and hemicellulosic fractions of the total cell polymers, and were thus not confined to anyone particular polymer. The D-galactose: L-galactose ratio in spinach polymers was ca. 70: 1.
Key words: Spinacia oleracea, Rosa sp. (<
© 1988 by Gustav Fischer Verlag, Stuttgart
curs exclusively as non-reducing termini; maize «fibre» hemicellulose contains the sequence L-Galp-(1-.4)-D-Xylp-(1 ....2)-LAraf(I-. ... ) linked to a xylan backbone (Whistler and Corbett, 1955). Nothing is known about the linkage of L-Gal in the primary cell wall. The biological role of L-Gal, if any, is completely unknown. However, an enzyme has been described which synthesises GDP-L-Gal from GDP-D-Man by epimerisation at C-3 and C-5 and this suggested that [3H]mannose could be a specific precursor with which to label L-Gal residues in vivo. Interest in the polymers of the primary cell wall has recently been fuelled by the discovery that certain domains of wall polysaccharides, when released in the form of diffusible oligosaccharides, possess biological activities (McNeil et al., 1984). The wish to investigate the natural occurrence (Fry, 1986), transport (Baydoun and Fry, 1985) and turnover (Bay-
L-Galactose in Primary Walls
doun and Fry, 1988) of such regulatory molecules, and to define their chemical structures, has prompted efforts to develop highly sensitive methods for their detection and characterisation. By far the most sensitive technique available is radioactive labelling of living cells followed by detection of wall components by scintillation-counting. Labelling can be either non·specific, where the precursor (e.g. D-[U-1 4C]glucose) is incorporated into all residues of the wall polymers, or specific, where the precursor is incorporated only into certain specified residues (Fry, 1988). Nonspecific labelling has the advantage that all organic wall components are made detectable, whether or not their presence was expected. Specific labelling has the advantage that fewer analytical steps are required to identify a particular residue; this can be valuable when the residues of interest are present in small quantities. Precursors for specific labelling include Lfucose [labels L-Fuc residues only (Roberts, 1968)], L-arabinose [labels L-Ara and D-Xyl residues (Neish, 1958; Fry and Northcote, 1983)] and uronic acids or myo-inositol [label DGalA, D-GlcA, D-Api, D-Xyl and L-Ara residues (Neish, 1958; see also Fry, 1985)]. Little use has been made of radioactive D-mannose as a precursor for wall polysaccharides. Roberts (1971) reported that in maize roots D-[U-14C]mannose was incorporated into DMan, L-Fuc and L-Gal, but not D-Glc, D-Gal, D-Xyl or L-Ara residues. However, in our preliminary experiments with cell cultures, [U-1 4C]mannose and [PH]mannose gave nonspecific labelling. [2-3H]Mannose has ben applied to the specific labelling of mannose residues in glycoproteins since the 3H is lost as 3H20 if the molecule takes the isomerase branch of the following pathway, but retained if it takes the branch leading to [3H]glycoprotein: D-[2- 3H]Mannose ~
D-[2- 3H]Mannose-6-P
Isomerase
D-Fructose-6-P (non-radioactive)
+ D-[2- 3H]Mannose-1-P
+
general cell metabolism
+
GDP-n-[2- 3H]Mannose
+ [3H]Glycoproteins
Materials and Methods Radioisotopes and cell cultures n-[2- 3H]Mannose (specific activity = 16 Ci/mmol) was from Amersham International pic, Amersham, Bucks. Its radiochemical purity was checked by PC in EAWand found to be 99.6 %. Cell suspension cultures of spinach (Spinacia oleracea L.) and rose (Rosa sp., cv. «Paul's Scarlet») were as used by Fry and Street (1980). Cell cultures of maize (Zea mays, Black Mexican sweetcorn) were maintained as described for rose. The [2-3H]mannose solution was supplied to the cultures, at 1-10 /LCi/ ml, without non-radioactive carrier (i.e., 0.6 - 6.0 /LM), for '" 4 h. For the collection of culture «filtrate», the cells were allowed to sediment under gravity for a few minutes; duplicate samples of the culture medium were mixed immediately with 10 volumes of the Triton scintillant, and two further samples were dried in vacuo, redissolved in the original volume of water, and then mixed with 10 volumes of Triton scintillant. Preparation ofpolymers To obtain total radioactive polymer, the cells were plunged into 4 volumes of absolute ethanol, solid D-mannose was added to 0.5 % (w/v), and the cells were washed with 80% (v/v) ethanol until the washings were no longer radioactive. The cells were finally washed with acetone and dried. Some polymer samples were fractionated as follows: 10 mg polymer was stirred with 5 ml AW at 25°C for 16 h, and the residue was washed with a further 2 x 3-ml portions of AW. To the pooled AW-extracts, 0.05 volumes of 10 % (w/v) ammonium formate and 5 volumes of acetone were added; the mixture was stored at 4 °C for 16 h. The precipitate which formed was washed in acetone and dried «
Hydrolysis
We report here that [2-3H]mannose is also useful for specific labelling of wall polysaccharides, the 3H being incorporated into D-Man, L-Fuc and L-Gal residues: GDP-D-[2-3H]Mannose
485
-----++
GDP-L-[3H]Fucose
For analysis of monosaccharide compOSition, polymers were usually hydrolysed in 2 M TF A at 120°C for 1 h. The TF A was removed in vacuo, and the residue analysed by Pc. For enzymic hydrolysis, 10 mg of [3H]polymer was shaken at 25°C for 16h in 1 ml of 1 % «Driselase» [a preparation from frpex lacteus containing numerous exo- and endo-glycosidases; (NH4)2S04-precipitated and de-salted (Fry, 1982)] at pH 4.5. Solubilised products ( > 95 % of the total 3H) were analysed by GPc.
+ GDP-L-[3H]Galactose
This enables a very sensitive and specific method to be applied to the study of L-Gal residues in the primary cell wall.
Galactose dehydrogenase To distinguish D- and L-[3H]galactose, the eH]galactose was purified by PC in BA W and EPW and incubated at 25°C for 16 h in 140/Ll of a solution containing 0.5 mM non-radioactive D-galactose, 2.0mM ,6-NAD+, 20mM Tris-acetate (pH 8.6), and 25 Units/ml D-
486
ELIAS A.-H. BAYDOUN and STEPHEN C. FRY
galactose dehydrogenase (from recombinant E. coli using the Pseudo· monas fluorescens gene; 1 Unit will oxidise Il'mol/min; Sigma Chemical Co. product no G-6637). The products were analysed by PC in EPW.
4
I"
E
'"'0
Chromatography PC was on Whatman no. 1, by the descending method in one of 5 solvents (BAW, BEW, EAW, EPW or ~W - see abbreviations). Peaks of 3H were located by scintillation-counting of strips (1 x 4 cm). Co-chromatography of 3H with internal markers was verified by recovery of the radioactive strips from the scintillant, washing in toluene, drying, and staining the non-radioactive marker. Monosaccharides were stained with aniline hydrogen phthalate, and aldonic acids with AgN0 3/NaOH (Fry, 1988). GPC was on a 45 x 1.5 cm column of Bio-Gel P-2, eluted with acetic acid/pyridine/water (1: 1: 23, v/v/v), pH 4.5. Internal markers of Blue Dextran and D-mannose were used.
Assay 0/ radioactivity Scintillants were 0.5 % PPO/O.OS % POPOP in toluene (for PC strips) and 0.33 % PPO/0.033 % POPOP in toluene/Triton XI00 (2: 1, v/v) (for aqueous samples). [3H]Polymers were hydrolysed in 2M TFA prior to assay for radioactivity.
x
3
2
E
Co <..l
J:
C')
Time(min)
Fig. 2: Uptake and conversion to 3H2 0 of [2- 3H]mannose (2.6I'Ci/ ml, added at time 0) by spinach cells growing with 2 % glycerol [circles] or 2 % glucose [squares] as sole carbon source. Total 3H (open symbols) and non-volatile 3H (solid symbols) in the culture filtrate was measured.
Results
Uptake and fate of[2_3H]Mannose [2-3H]Mannose (0.6 t-tM) rapidly started to disappear from the medium of glycerol-grown spinach cells (Fig. 1). After a short time (10-20 min), 3H started to reappear in the medium. This was shown, from its disappearance on drying, to be largely 3H20. The [2-3H]mannose had been 50 % taken up and/or processed to 3H2 0 within 4-8 min (Fig. 1). Conversion of [2-3H]mannose to 3H20 was shown to be a cellular process by the fact that prolonged incubation of [2-3H]mannose in freshly-collected culture-filtrate did not produce any 3H2 0. This confirmed that mannose was indeed taken up and metabolised remarkably quickly. Spinach cells grown on sucrose, glucose or glycerol showed similar results, but uptake was slower in the (slowergrowing) glycerol cells (Fig. 2). This implies that D-mannose was taken up and phosphorylated by a system that is not swamped by ca. 20 - 56 mM D-glucose, the 2-epimer of Dmannose. This is surprising in view of the fact that D-glucose will protect cells from the toxic effects of excess D-mannose (Goldsworthy and Street, 1965). Results similar to those shown in Fig. 2 were obtained with cultured cells of rose and matze. In addition to 3H20, some cellular [3H]polymer was produced (ca. 10% of the supplied [2-3H]mannose after 125 min). Ca. 1 % of the radioactive material reappearing in the culture medium was also shown by PC in EAW to be polymer (RF 0.00).
1.0
E
Co <..l
J: 0.5
C')
o~--------~---------+--------~ o 20 40 60 Time(min)
Fig. 1: Uptake of [2-3H]mannose (1I'Ci/ml, added at time 0) by spinach cells growing with 2 % glycerol as sole carbon source. The graph plots the amount of 3H recovered in the culture filtrate: 0 - - 0 = total 3H; e--e = non-volatile 3H; 1::,.--1::,. = 3H20 (by difference).
Monomer composition offH]polymers [3H]Polymer from glucose-grown spinach cells was dried, hydrolysed in 2M TFA, and analysed by PC in EPW. Five peaks of 3H were reproducibly found (Fig. 3) co-migrating with polymer, galactose, mannose, fucose and 2-O-methylfucose respectively. The putative galactose spot was confirm-
L-Galactose in Primary Walls Xyl
I-----<
GaiA
2.0
'E"
~
1.5
Gal
Gle
Ara
............ I-----I~........-......
Fue ~
487
Rha
I------t
Rib
1-----1
Man
D-Man
L-Fue
C')
'~
Fig. 3: Paper chromatography of acid-hydrolysate of total [3H]polymers from glucose-grown spinach cells fed [2-3H]mannose (10j.tCi/ml) for 4h. Solvent: EPW. The bars indicate the positions of authentic markers. Imm = chromatographically-immobile material.
1·0
)(
E
c.
.8. Imm :t 0·5
C')
Me-L-Fue?
0~-=~=-----~---=~----~~-=-----3~0~---------4~0==~~
ed as such by co-chromatography with authentic L-galactose in BAW, BEW and c/>W. To determine whether the [3H]galactose was D- or L-, a sample was purified by PC and incubated with D-galactose dehydrogenase in the presence of non-radioactive D-galactose and excess NAD+. PC of the products in EPW showed that the non-radioactive D-galactose had been completely converted to galactonic acid (RCal = 0.1- 0.2), but all the 3H still co-chromatographed with galactose, indicating that the pH]galactose was 100 % L-galactose. This agrees with the expected range of sugar interconversions described in the Introduction. The Man and Fuc were not analysed in detail, but are assumed to be D-Man and L-Fuc respectively (Roberts, 1971). The material with RFO.OO (Fig. 3) was re-hydrolysed (2M TFA at 120°C for 1 h; or 4 M TF A at 120°C for 2 h): this caused slight (5 - 8 %) hydrolysis to mannose, but the bulk of the material was essentially acid-resistant, and is unidentified.
Kinetics of labelling of the polymer components Spinach cells were incubated with [2-3H]mannose and samples of the pH]polymer were analysed at intervals. DMan and L-Gal residues started to be labelled with a lag of < 5 min (Fig. 4). D-Man residues ceased to accumulate further 3H after 1 h incubation. However, the L-Gal residues continued to accumulate 3H well after 1 h. The fact that L-Gal residues started to be labelled with a negligible lag, and linearly for ca. 20 min, suggests that the endogenous pool of non-radioactive GDP-L-galactose was rapidly turning over; we therefore cannot explain the continued labelling of L-Gal residues from 1- 3 h by slow turnover of the GDP-L-[2- 3H]galactose pool. An alternative explanation is that the D-Man -+ L-Gal conversion occurs after incorporation into polymer(s), as reported for the formation of L-guluronic acid residues by 5-epimerisation of polysaccharide-bound D-mannuronic acid ~esidues (Haug and Larsen, 1969). This requires further testmg. L-Fuc residues were labelled at a linear rate after a slightly longer lag (9 min), presumably reflecting the time taken to convert GDP-D-Man into GDP-L-Fuc. The unidentified ma-
em from origin
terial of RF 0.00 was labelled without a detectable lag, but then lost ca. 65 % of its 3H, indicating turnover of a portion of this material.
Graded acid hydrolysis of the L-Gal residues To discover whether the L-Gal residues were mainly in the pyranose or the more acid-labile furanose form, graded acidhydrolysis was performed. Aliquots of [3H]polymer were incubated at 120°C for 1 h at various pHs, and the products analysed by PC in EPW. L-Gal residues were released as monosaccharides at a rate intermediate between D-Man and L-Fuc (Fig. 5), both of which are known to occur as pyranosides. If the L-Gal residues are non-reducing termini, as suggested for other systems (Whistler and Corbett, 1955; Bretting et aI., 1985), our data show that L-Gal was pyranosidically linked. Furanosides would have been released by milder acid [see data for sucrose: Fig. 5].
Products ofDriselase digestion «Driselase» has proved valuable in the characterisation of sugar linkages in the plant cell wall (Fry, 1986, 1988). It converts all the major sugar components of the spinach cell wall to monosaccharides except for the a-Xyl residues of xyloglucan, which are released as the disaccharide xylosyl-a(1-+6)-glucose. Driselase contains little protease and esterase activity. Nothing was known about the susceptibility of LGal residues, or the polymers that bear them, to Driselase. Therefore, Driselase digestion products of [3H]polymer were fractionated by GPC on Bio-Gel P-2. Each fraction was then hydrolysed with 2 M TF A to reveal the [3H]monosaccharides present (Fig. 6). L-Gal residues were less effectively released than D-Man or L-Fuc, which were largely converted to monosaccharide. About 43 % of the L-Gal was recovered in the tri- to tetrasaccharide zone [d. elution-volume of maltotetraose (Fig. 6)], showing that Driselase was poor in L-galactosidase activity. Recovery of D-Man and L-Fuc in this zone was 9 and 7 % respectively. Only 10 % of the L-Gal was recovered in the totally-excluded (high-M r ) fractions, in contrast to D-Man and L-Fuc (31 and 20 % respectively). This in-
488
ELIAS
A.-H. BAYDOUN and STEPHEN C. FRY
Man
75 32 .
;.,
0
E 50 :J E
x
CIl
E
'0 25
'-
E
o~~----~----~--~
o
N
I
0
x
3
10
Fig. 5: Graded acid hydrolysis of eH]polymers obtained by feeding [2- 3H]mannose (lO/-lCi/ml) to glucose-grown spinach cells for 4h. Aliquots of the [3H]polymer were hydrolysed for 1 h at 120°C in various acid solutions; products were analysed as in Fig. 3. The yield of glucose + fructose from non-radioactive sucrose (Suer) is included for comparison. Imm = chromatographically-immobile material.
E
c.
0
J:
C')
2
Approx. pH
5
o~--
_____________________
10
Imm
L·Cal: D-Cal ratio in spinach polymers
5
o
2
distributed between all the other fractions (except a-cellulose), indicating that L-Gal residues are not confined to any one particular polymer.
3
Time (h)
Fig.4: Time course for incorporation of 3H from [2-3H]mannose (10/-lCi/ml) into four components of polymers by cells growing with 2 % glycerol as sole carbon source. The [2-3H]mannose was fed at time 0, and polymer samples were taken at intervals for analysis as shown in Fig. 3. Imm = chromatographically-immobile material.
Hydrolysis of non-radioactive spinach cells in TF A, followed by 2-dimensional PC in EPW and BAWand staining with aniline hydrogen phthalate, indicated a ratio of total galactose: fucose of ca. 10: 1. The data in Fig. 3 indicate a ratio of L-Gal: Fuc of 1 : 7. Therefore the ratio L-Gal: D-Gal was ca. 1: 70, or 1.4 % L-Gal. This value is considerably lower than that (12 %) reported for sycamore cells (Roberts and Harrer, 1973), but nevertheless confirms that a significant minority of the Gal residues are the L-enantiomer.
Discussion dicates that the «core» of the polymer that bears the L-Gal was highly susceptible to Driselase digestion and thus unlikely to be a protein.
Fractionation of L-galactose-containing polymers To obtain more information about the polymers that bear L-Gal residues, the [3H]polymers were fractionated. The partition of L-Gal residues between the fractions is compared with that of D-Man and L-Fuc (Table 1). As expected, much of the D-Man and L-Fuc was in glycoprotein. In contrast, much of the L-Gal was pectic; however, it was also broadly
D-Mannose is toxic at high concentrations, inhibiting growth and respiration and promoting starch synthesis, probably by sequestering much of the cell's phosphate supply as Man-6-P (Goldsworthy and Street, 1965; Chen-she et aI., 1975). This suggests that plant cells have little Man-6-P isomerase activity [see scheme in Introduction], a view supported by Roberts' (1971) observation that, in maize roots, D{U- 14C]mannose is not readily incorporated into derivatives of Fru-6-P [i.e. polysaccharide-bound D-Glc etc.]' In contrast, our results with cultured cells indicate that tracer levels of Man-6-P were isomerised very rapidly indeed: thus, D-[U-14C]mannose and D{PH]mannose were extensively
L-Galactose in Primary Walls
489
Table 1: Distribution of L-[3H]Gal, D-[3H]Man and L-eH]Fuc residues between various cellular polymer fractions. L-Gal D-Man L-Fuc kcpm*) % kcpm*) % kcpm*) % .pAW-soluble 21 47 141 43 18.7 565 Cold H 20 soluble 14.4 16 76 6 23 7 37.3 41 365 31 80 24 Pectin Hemicellulose 18.9 21 180 15 81 25 a-Cellulose 1.0 1.1 4 0.3 3 0.9
Fraction
30
Total (10 mg)
Man
*) kcpm
SO
E
100
1200
100
328
100
thousands of counts per minute
~ these three quantitatively minor residues of the wall polysaccharides.
til
-
=
90.3
20
0
Acknowledgements
;!1
Weare most grateful to Miss Sheenagh Bryson for excellent technical assistance. E. A.-H. B. thanks Yarmouk University, Irbid, Jordan for a research grant. S. C. F. thanks the EEC for the award of a research grant under their «Biotechnology Action Programme». 10
References
o~~
____________
0.25
~
__________
0.5 Column
volumes
~
___
0.75 eluted
Fig. 6: GPC on Bio-Gel P-2 of Driselase-digested [3H]polymers obtained by feeding [2- 3H]mannose (10 "Ci/ml) to glucose-grown spinach cells for 4 h. Each Bio-Gel fraction was acid-hydrolysed and analysed as in Fig. 3. Imm = chromatographically-immobile material. BD
I=
Internal marker Blue Dextran.
f--~M""---II
=
Internal marker D-mannose.
f----,M'-'.4-'---I1
=
Position of elution of maltotetraose (run under identical conditions but on a separate occasion) relative to Blue Dextran and D-mannose.
metabolised to derivatives of Fru-6-P, and 0-[2- 3H]mannose quickly lost ca. 90 % of its 3H as 3H2 0. Since we tested the [23H]mannose at low concentrations ( =:::: 6 I'M), our results do not say whether Man-6-P isomerase activity had a high V max. Nevertheless, the cultured cells were clearly able to mobilise the levels of Man-6-P formed from (non-toxic) tracer levels of exogenous mannose. Our results show that for carrier-free 0-[3H]mannose to be useful as a specific precursor of o-Man, L-Fuc and L-Gal residues, it must carry the label in the 2-position. Although only 10 % of the 3H is recruited into polymers, the rapidity of uptake and specificity of incorporation makes 0-[23H]mannose a valuable precursor for the investigation of
ANDERSON, E.: The preparation of I-galactose from flaxseed mucilage.]. BioI. Chern. 100, 249-253 (1933). BAYDOUN, E. A.-H. and S. C. FRY: The immobility of pectic substances in injured tomato leaves and its bearing on the identity of the wound hormone. Planta 165, 269-276 (1985). - - The in vivo turnover of xyloglucan nonasaccharide, a possible biologically active cell wall fragment. (In preparation) (1988). BRETTING, H., G. JACOBS, I. BENECKE, W. A. KONIG, and J. THIEM: The occurrence of L-galactose in snail galactans. Carbohydr. Res. 139,225-236 (1985). CHEN-SHE, S., D. H. LEWIS, and D. A. WALKER: Stimulation of photosynthetic starch formation by sequestration of cytoplasmic orthophosphate. New Phytol. 74, 383 -392 (1975). FRY, S. c.: Phenolic components of the primary cell wall. Feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharides. Biochem. J. 203, 493 - 504 (1982). - Primary cell wall metabolism. Oxford Surv. Plant Mol. Cell BioI. 2,1-42 (1985). - In-vivo formation of xyloglucan nonasaccharide: a possible biologically active cell-wall fragment. Planta 169, 443-453 (1986). - The Growing Plant Cell Wall: Chemical and Metabolic Analysis. 333 pp. Longmans, London (1988). FRY, S. C. and D. H. NORTHCOTE: Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides. Plant Physiol. 73, 1055-1061 (1983). FRY, S. C. and H. E. STREET: Gibberellin-sensitive suspension cultures. Plant Physiol. 65, 472-477 (1980). GOLDSWORTHY, A. and H. E. STREET: Carbohydrate nutrition of tomato roots. VIII. The mechanism of the inhibition by D-mannose of the respiration of excised roots. Ann. Bot., N. S. 29, 45-58 (1965). HAUG, A. and B. LARSEN: Biosynthesis of alginate. Epimerisation of D-mannuronic acid to L-guluronic acid residues in the polysaccharide chain. Biochim. Biophys. Acta 192,557 -559 (1969). McNEIL, M., A. G. DARVILL, S. C. FRY, and P. ALBERSHEIM: Structure and function of the primary cell walls of plants. Annu. Rev. Biochem. 53, 625-663 (1984).
490
EUAS A.-H. BAYDOUN and STEPHEN C. FRY
NEISH, A. c.: The biosynthesis of cell wall carbohydrates. IV. Further studies on cellulose and xylan in wheat. Can. J. Biochem. Physio1.36, 187 -193 (1958). ROBERTS, R. M.: The metabolism of L-fucose by sycamore (Acerpseudoplatanus L.) cell cultures. Arch. Biochem. Biophys. 128, 818-820 (1968). - The metabolism of D-mannose- 14C to polysaccharide in corn roots. Specific labeling of L-galactose, D-mannose, and L-fucose. Arch. Biochem. Biophys. 145, 685-692 (1971).
ROBERTS, R. M. and E. HARRER: Determination of L-galactose in polysaccharide material. Phytochemistry 12, 2679-2682 (1973). WHISTLER, R. L. and W. M. CORBETT: Oligosaccharides from partial acid hydrolysis of corn fiber hemicellulose. J. Am. Chern. Soc. 77,6328-6330 (1955). WINTERSTEIN, E.: Ueber die aus Chagual-Gummi entstehenden Glucosen (inactive Galaktose und Xylose). Ber. deut. Chern. Ges.31, 1571-1573 (1898).