©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Trehalose as a Possible Precursor of the Sulfated

L

-Galactan in the Ascidian Tunic (*)

(Received for publication, October 12, 1994)

Paulo A. S. Mourão (§) Ana-Maria S. Assreuy

From the Departamento de Bioquímica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS AND FUTURE PERSPECTIVES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Among sulfated polysaccharides, those in the tunic of ascidians are unique: their major constituent sugar is galactose, which occurs exclusively in the L-enantiomeric form. Incorporation of D-[^14C]glucose into tunic slices in vitro revealed that the cells epimerize D-glucose into L-galactose during biosynthesis of the sulfated polysaccharides. The interconversion of these two sugars involves exchange of hydrogen atoms at the epimerization sites with protons of the medium. Tunic cells also synthesize trehalose, although this disaccharide is not a prominent constituent of the tissue. Pulse-chase experiments using D-[^14C]glucose reveal that incorporation of label into trehalose precedes the synthesis of the sulfated L-galactan. In addition, the loss of label from trehalose coincides with the appearance of label in the sulfated L-galactan. Based on these results, we speculate that trehalose in the ascidian tunic may be a precursor of the sulfated L-galactan.


INTRODUCTION

Sulfated polysaccharides from different invertebrate tissues include several that are not found in vertebrates. Novel sulfated polysaccharides have been reported in the tunic of ascidians(1, 2, 3, 4, 5, 6, 7) , the body wall of a sea cucumber(8, 9, 10, 11, 12) , and the jelly coat from sea urchin eggs(13) .

The sulfated polysaccharides in the tunic of ascidians (Chordata, Tunicata) occur as fractions that are distinct in molecular weight and chemical composition. A high molecular weight glycan composed mainly of sulfated galactose residues is the preponderant fraction in all ascidians studied so far(2, 3, 4, 5, 6, 7) .

In previous studies, we showed that the galactose from the sulfated polysaccharides of ascidians occurs entirely in the L-enantiomeric form(3, 7, 14) . Although L-galactose is a constituent of several polysaccharides (15, 16, 17, 18) , these were the first reports of sulfated polysaccharides containing high amounts of L-galactose but no D-galactose. In the high molecular weight fraction, galactose occurs mainly as alpha-L-galactopyranosides, linked glycosidically through positions 1-4 and sulfated at position 3 (3, 4, 5, 6, 7) . The small amounts of glucose units found in these polysaccharides from some species of ascidians are the D-isomer, primarily in beta-D-glucopyranosyl units(3, 4) .

The pathway involved in the incorporation of L-galactose into polysaccharides is not known. The major obstacle for this study is that L-galactose occurs in the presence of large amounts of the D-isomer of this sugar(15, 16, 17, 18) . It occurred to us that the ascidian tunic could be useful for studies on the metabolism of L-galactose.

In a previous study we reported that the biosynthesis of sulfated polysaccharides from radiolabeled precursors is more intense in the region of the ascidian tunic containing epidermal cells and that subsequently the incorporated material is redistributed into more peripheral areas of the tunic(19) . Surprisingly, the most effective precursor was D-glucose(20) .

We now investigate in detail the epimerization of D-glucose to L-galactose during the biosynthesis of the sulfated L-galactan in the ascidian tunic. Our results reveal an unexpected parallel between the biosynthesis of sulfated L-galactan and the incorporation of D-[^14C]glucose into trehalose in the ascidian tissue.


EXPERIMENTAL PROCEDURES

Materials

D-[U-^14C]Glucose, D-[2-^3H]glucose, D-[3-^3H]glucose, D-[5-^3H]glucose, and D-[6-^3H]glucose were purchased from DuPont NEN, and [^3H]H(2)O (2.02 mCi/ml) was from Amersham. Chondroitin 6-sulfate from shark cartilage, Dactylium dendroidesD-galactose oxidase, porcine kidney trehalase, bakers' yeast alpha-D-glucosidase, almond beta-D-glucosidase and twice crystallized papain (type IV) were from Sigma. 1,9-Dimethylmethylene blue was from Aldrich, and agarose (standard low M(r)) was from Bio-Rad. [^14C]Trehalose was prepared from D-[U-^14C]glucose, using a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted (21) .

Incorporation of ^14C- or ^3H-Labeled Sugar into the Ascidian Tunic

The ascidian Styela plicata was collected in Guanabara Bay, Rio de Janeiro, and transported to the laboratory immersed in sea water. Thereafter, the ascidian was cut longitudinally, the viscera were removed, and the tunic was cut transversely into slices approximately 1 mm thick. These slices were immersed immediately in the incubation medium containing 423 mM NaCl, 9 mM KCl, 9 mM Na(2)S0(4), 23 mM MgCl(2), 9 mM CaCl(2), and 2 mM NaHC0(3), and washed three times with 5 ml of this solution. Approximately 15 mg (wet weight) of the slices was incubated for different times at 20 °C either with 0.7 µCi (1.05 nmol) of the ^14C-labeled sugar or with 20 µCi (0.66 nmol) of the ^3H-labeled sugar in 100 µl of the incubation medium. At the end of the labeling period, the medium was decanted, and the tunic slices were washed five times with 5 ml of the incubation medium. In some experiments the ascidian tissue was incubated with D-[U-^14C]glucose for 6 h, then the incubation medium was removed, and a new medium without D-[U-^14C]glucose was added to the tissue slices and incubated for an additional period of time. The slices were then immersed in 2 ml of acetone, where they were kept for 24 h at 4 °C and then dried at 80 °C for 60 min.

The ^14C- and ^3H-labeled compounds were isolated following the method described previously for fresh tissue(2) . Briefly, the dried slices (2.0 mg, dry weight) were suspended in 0.5 ml of 0.1 M sodium acetate buffer (pH 5.0) containing 30 µg of twice crystallized papain, 5 mM EDTA, and 5 mM cysteine and incubated at 60 °C for 24 h. The incubation mixture was then centrifuged (2,000 times g for 10 min at room temperature), and the incubation of the pellet with papain was repeated. The clear supernatants were combined and mixed with 2 volumes of absolute ethanol. The precipitate formed after standing at -10 °C for 24 h was collected by centrifugation (2,000 times g for 15 min at room temperature), dried at 80 °C for 1 h, and dissolved in 100 µl of distilled water.

To estimate the amount of radioactivity transferred from ^14C- or ^3H-labeled sugars to other molecules of the ascidian tunic, the solution described above was applied to Whatman 3MM chromatographic paper and developed in 1-butanol/pyridine/water (3:2:1, v/v) for 24 h. The origin, now free of ^14C- or ^3H-labeled monosaccharides, was cut out, added to 5 ml of 0.5% PPO(^1)/toluene solution, and counted in a liquid scintillation counter. Throughout the text we refer to this result as ``incorporated radioactivity.''

Identification of the ^14C-Labeled Polysaccharides by Agarose Gel Electrophoresis

The origin of the chromatogram described above was removed from the PPO/toluene solution, washed three times with 10 ml of acetone, and dried. Thereafter, the ^14C-labeled compounds were eluted from the paper with distilled water, dried on a rotary evaporator, and dissolved in 100 µl of distilled water. The sulfated polysaccharides in the solution were analyzed by agarose gel electrophoresis, as described previously(2) . Briefly, 20 µl of this solution was applied to an agarose gel (0.5%, w/v) and run in 0.05 M 1,3-diaminopropane/acetate buffer (pH 9.0) for 1 h at 120 V. The sulfated polysaccharides in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide in water and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v). After staining, the gel was washed for about 15 min in acetic acid/ethanol/water (0.1:5:5, v/v). The ^14C-labeled polysaccharides were visualized by autoradiography of the stained gels.

Identification of the ^14C- or ^3H-Labeled Sugar Residues Incorporated into the Ascidian Tunic

Eighty microliters of the solution of intact ^14C- or ^3H-labeled compounds, obtained by elution from the origin of the chromatogram (see above), were subjected to acid hydrolysis (6.0 N trifluoroacetic acid at 100 °C for 4 h). The resulting mixture of labeled sugars was applied to Whatman No. 1 paper and separated by descending chromatography in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h. The chromatogram was cut transversely into 3-mm-wide strips, which were added to 5 ml of 0.5% PPO/toluene solution and counted in a scintillation counter. A longitudinal strip showing migration of a standard solution containing 10 µg each of D-galactose, D-glucose, and D-mannose was developed with silver nitrate.

Purification of the ^14C-Labeled Molecules Extracted from Ascidian Tunic

Preparation of Large Amounts of ^14C-Labeled Molecules

Slices of ascidian tunic (120 mg, wet weight) were incubated at 20 °C with 3.5 µCi (5.25 nmol) of D-[U-^14C]glucose in 500 µl of incubation medium (see above) for 3 or 6 h. In some experiments the ascidian tissue was incubated with D-[U-^14C]glucose for 6 h, then the incubation medium was removed, and a new medium without D-[U-^14C]glucose was added to the tissue slices and incubated for an additional 3 h. At the end of the pulse and chase period, the medium was decanted, and the tissue slices were washed five times with the incubation medium, then immersed in acetone, and the ^14C-labeled molecules were extracted from the tissue with papain, precipitated with ethanol, and dissolved in distilled water, as described above. In some experiments the same methodologies were applied to 45 mg (dry weight) of the ascidian tunic, but without the pulse with D-[U-^14C]glucose to obtain large amounts of nonlabeled material. In either case the aqueous solution was applied to Whatman 3MM chromatographic paper and developed in 1-butanol/pyridine/ water (3:2:1, v/v) for 24 h. The origin, now free of monosaccharides, was cut out, and the compounds were eluted with distilled water, dried in a rotary evaporator, and dissolved in 1 ml of distilled water.

Anion Exchange Chromatography

The above solution was applied to a column (10.0 times 1.0 cm or 1.0 times 1.0 cm) of DEAE-cellulose preequilibrated with 0.05 M pyridine/acetate buffer (pH 6.0) and developed with a gradient of NaCl, as described in the figure legends. The fractions were checked by the metachromatic assay (22) and by the Dubois reaction(23) ; the radioactivity was counted on a scintillation counter, and the NaCl concentration was estimated by conductivity. The fractions containing the sulfated polysaccharides were pooled, dialyzed against distilled water, and lyophilized. The fractions containing the nonacidic compound (see Fig. 5and Fig. 7) were concentrated on a rotary evaporator without previous dialysis.



Figure 5: Separation of the ^14C-labeled molecules extracted from the ascidian tunic by anion exchange chromatography on DEAE-cellulose. Panels A-C, slices of ascidian tunic (120 mg, wet weight) were incubated at 20 °C with 3.5 µCi (5.25 nmol) of D-[U-^14C]glucose in 0.5 ml of the incubation medium (see ``Experimental Procedures'') for 3 (panel A) or 6 h (panel B). In panel C, the ascidian tissue was incubated with D-[U-^14C]glucose for 6 h (as in panel B), but then the incubation medium was removed, and a new medium without D-[U-^14C]glucose was added to the tissue slices and incubated for additional 3 h. At the end of the incubation period, the ^14C-labeled molecules were extracted from the tunic slices by papain digestion, precipitated with ethanol, and dissolved in distilled water, as described under ``Experimental Procedures.'' The solutions of ^14C-labeled molecules were applied to Whatman 3MM chromatographic paper and developed in 1-butanol/pyridine/water (3:2:1, v/v) for 24 h. The origins, now free of ^14C-labeled monosaccharides but containing the incorporated radioactivity, were cut out, and the ^14C-labeled compounds were eluted with distilled water, dried in a rotary evaporator, and dissolved in 0.5 ml of distilled water. This solution was applied to a column (1 times 1 cm) of DEAE-cellulose preequilibrated with 0.05 M pyridine/acetate buffer (pH 6.0), washed with 15 ml of the same buffer, and developed with a linear gradient of NaCl, prepared by mixing 15 ml of 0.05 M pyridine/acetate buffer (pH 6.0) with 15 ml of 1.5 M NaCl in the same buffer, at a flow rate of 12 ml/h. Fractions were collected each 2 min and checked by the metachromaticity assay (circle), the radioactivity was counted in a liquid scintillation counter (bullet), and the NaCl concentration(- - -) was estimated by conductivity. The fractions containing the sulfated polysaccharides (horizontal bars) were pooled, dialyzed against distilled water, and lyophilized. The fractions containing the nonacidic compound were pooled and concentrated on a rotary evaporator, without previous dialysis. Panel D shows the absolute amounts of label incorporated into the nonacidic compound (circle) and into the sulfated polysaccharides (bullet) during the pulse-chase experiment.




Figure 7: Purification of the nonacidic compound. Slices of ascidian tunic (120 mg, wet weight) were incubated with 3.5 µCi (5.25 nmol) of D-[U-^14C]glucose in 0.5 ml of the incubation medium. The ^14C-labeled compounds were extracted and separated from ^14C-labeled monosaccharides as described in the legend of Fig. 5and under ``Experimental Procedures.'' The same procedures were applied to 46 mg (dry weight) of ascidian tunic, but using nonradioactive glucose. The origins of the chromatograms of both labeled and unlabeled compounds were eluted with distilled water, combined, concentrated on a rotary evaporator, and dissolved in 1.0 ml of distilled water. In panel A this solution was applied to a column (10 times 1 cm) of DEAE-cellulose preequilibrated with 0.05 M pyridine/acetate buffer (pH 6.0), followed by 30 ml of the same buffer, then 30 ml of 2.0 M NaCl (arrow), at a flow rate of 8 ml/h. Fractions (1.1 ml) were collected and checked by the reaction of Dubois et al. (circle) and by the metachromatic assay (times), and counted (bullet) in a liquid scintillation counter. In panel B, the fractions containing the nonacidic compound (pool 1 from panel A) were concentrated on a rotary evaporator, dissolved in 1 ml of distilled water, and applied to a 87.0 times 1.5-cm column of Bio-Gel P-4 (400 mesh). Fractions were eluted and analyzed as described for panel A, and those containing the nonacidic compound (bracket) were pooled and concentrated on a rotary evaporator. The column was calibrated using chondroitin 6-sulfate from shark cartilage, cellobiose, and D-glucose (arrows 1-3, respectively).



Gel Chromatography of the Nonacidic Compound

The ^14C-labeled nonacidic compound, which was not retained on the anion exchange column (see above), was applied to a 87.0 times 1.5-cm column of Bio-Gel P-4 (400 mesh), eluted at a flow rate of 8 ml/h with 0.05 M pyridine/acetate buffer (pH 6.0). Fractions were collected each 8 min and checked by the metachromatic assay (22) and by the Dubois reaction (23) and counted on a liquid scintillation counter. The V(0) and V(t) of the column were determined by the elution of blue dextran and cresol red, respectively. In addition, the column was calibrated using chondroitin 6-sulfate from shark cartilage (average molecular mass of 40 kDa), cellobiose, and D-glucose.

Enzymatic Degradation

Oxidation with D-Galactose Oxidase

The mixture of ^14C-labeled sugars obtained by acid hydrolysis of the ^14C-labeled compounds (2,000 cpm) and a mixture of standard sugars containing 10 µg each of D-galactose, D-glucose, and D-mannose were incubated with 1 unit of D. dendroidesD-galactose oxidase (24) in 50 µl of 0.05 M sodium acetate buffer (pH 7.0). After 4 h at 37 °C, the reaction mixtures were developed by descending chromatography on Whatman No. 1 paper in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h, and transverse strips 3 mm wide were counted as before. In addition, a longitudinal strip from the chromatogram was developed with silver nitrate for the identification of nonlabeled standard sugars, before and after incubation with D-galactose oxidase.

Incubation of the Nonacidic Compound with alpha-D-Glucosidase, beta-D-Glucosidase, and Trehalase

The ^14C-labeled nonacidic compound (2,000 cpm), after purification on anion exchange and gel chromatography columns (see above), was incubated with: (a) 0.2 unit of bakers' yeast alpha-D-glucosidase in 50 µl of 50 mM sodium acetate buffer (pH 5.0), or (b) 0.2 unit of almond beta-D-glucosidase in 50 µl of 50 mM sodium acetate buffer (pH 6.8), or (c) 0.2 unit of porcine kidney trehalase in 50 µl of 20 mM potassium acetate buffer (pH 6.5). After incubation at 37 °C for 4 h, the mixtures were applied to Whatman No. 1 paper and developed by descending chromatography in isobutyric acid, 1 N NH(4)OH (5:3, v/v) for 24 h. The chromatograms were cut transversely into 3-mm-wide strips, which were added to 5 ml of 0.5% PPO/toluene solution and counted in a scintillation counter.

To assess the alpha-D- and beta-D-glucosidase activities, the p-nitrophenol liberated from p-nitrophenyl alpha-D-glucopyranoside and from p-nitrophenyl beta-D-glucopyranoside, respectively, was quantified. The enzymes (0.2 unit) were incubated at 37 °C for 30 min in 50 µl of 50 mM sodium acetate buffer (pH 5.0 for alpha-D-glucosidase and pH 6.8 for beta-D-glucosidase). The reaction was terminated with 1 ml of 0.25 M glycine buffer (pH 10.0), and the absorbance was read at 540 nm. To assess trehalase activity, trehalose (50 µg) was incubated with the enzyme (0.1 unit) at 37 °C for 4 h in 50 µl of 20 mM potassium acetate buffer (pH 6.5). The mixture incubated with trehalase and the appropriate control solution were applied on Whatman No. 1 paper and developed by descending chromatography in isobutyric acid, 1 N NH(4)OH (5:3, v/v) for 24 h. The sugars were visualized by silver nitrate staining.


RESULTS AND DISCUSSION

Epimerization of D-Glucose to L-Galactose during the Biosynthesis of a Sulfated L-Galactan in the Ascidian Tunic

Upon incubation of the tunic slices with D-[U-^14C]glucose the incorporation of radioactivity begins immediately and increases steadily in the first 4 h up to a plateau value (Fig. 1A). To show whether the ^14C-labeled sugar was incorporated into sulfated polysaccharides, the glycans extracted from the tunic during the experiment of Fig. 1A were subjected to agarose gel electrophoresis (Fig. 1B). On an autoradiogram of the stained gel, the radioactive bands coincide with the two fractions of sulfated polysaccharides that have been characterized in previous studies(2, 3) . Both fractions (F-1 and F-2) are labeled.


Figure 1: Labeling of the ascidian polysaccharides with D-[U-^14C]glucose. Panel A, time course of incorporation of D-[U-^14C]glucose. Slices of ascidian tunic (15 mg, wet weight) were incubated at 20 °C with 0.7 µCi (1.05 nmol) of D-[U-^14C]glucose in 100 µl of the incubation medium (see ``Experimental Procedures''). At different times, the medium was decanted, and the slices were washed five times with the incubation medium, then immersed in 2 ml of acetone, kept for 24 h at 4 °C, and dried. The ^14C-labeled compounds were extracted by papain digestion, and the incorporated radioactivity was quantitated as described under ``Experimental Procedures.'' Panel B, agarose gel electrophoresis of the ^14C-labeled polysaccharides. Twenty microliters of the ascidian polysaccharides, labeled with D-[U-^14C]glucose for different periods of times, was applied to a 0.5% agarose gel, and electrophoresis was carried out in 0.05 M 1,3-diaminopropane/acetate buffer (pH 9.0) for 1 h at 120 V. The sulfated polysaccharides from the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide in water for 12 h and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v). The radioactive bands corresponding to the ^14C-labeled polysaccharides were detected by autoradiography of the fixed and stained gel. The electrophoretic migration of standard F-1 and F-2 fractions are indicated at the right. Panel C, identification of the ^14C-labeled sugars. The acid hydrolysates of the labeled compounds, before (bullet) and after (circle) incubation with D-galactose oxidase, were applied to Whatman No. 1 paper and chromatographed in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h. The chromatogram was cut into 3-mm-wide strips and counted in a liquid scintillation counter. The relative amounts (percentage) of label appearing in each of the sugars, on the basis of the area of each peak compared with total area, are shown beside each peak. The upper portion of the panel (a and b) shows a strip from a chromatogram developed with silver nitrate where a standard solution was applied, containing 10 µg each of D-galactose (lane 1), D-glucose (lane 2), and D-mannose (lane 3), before (a) and after (b) a 4-h incubation with D-galactose oxidase (origin at right). Incubation conditions and the hydrolysates are described under ``Experimental Procedures.''



Identification of the ^14C-labeled sugars in the acid hydrolysate of the ascidian polysaccharides shows a high proportion of label in the galactose residues (closed circles in Fig. 1C). The [^14C]galactose is totally resistant to oxidation by D-galactose oxidase (open circles in Fig. 1C), indicating that this sugar is entirely in the L-enantiomeric form, as already observed for the nonlabeled polysaccharides(3, 7, 14) . The measurement of the oxidation by D-galactose oxidase included a control, which shows the total oxidation of the D-galactose standard (b in Fig. 1C).

Overall, these results demonstrate that cells in the ascidian tunic incorporate D-[^14C]glucose and epimerize this sugar into L-[^14C]galactose during the biosynthesis of their constituent sulfated polysaccharides.

The [^3H]Hydrogen Atoms at Carbons 2, 3, and 5 of D-Glucose Are Exchanged with the Protons of Water during the Epimerization of D-Glucose to L-Galactose

Epimerization of D-glucose to L-galactose involves inversion of the configuration of carbon atoms 2, 3, and 5 of the hexosyl moieties, as shown for the beta-D-glucopyranosyl and alpha-L-galactopyranosyl configurations.

The interconversion of sugars is a relatively common reaction in carbohydrate metabolism. It can proceed as in the enzymatic conversion of UDP-D-glucose to UDP-D-galactose. The interconverting enzyme and all other such epimerases exhibit an absolute requirement for a tightly bound molecule of NAD or NADP. Oxidation is followed by reduction with inversion of the configuration at the epimerization site, and a proton is exchanged with the enzyme-bound NAD or NADP(25) . In contrast, in the epimerization of D-mannose to D-glucose, which involves the action of D-mannose phosphate isomerase, the hydrogen atom at carbon 2 of D-mannose is exchanged with the protons of water(26) . A similar mechanism occurs in several other epimerization reactions, such as the double epimerization in the synthesis of UDP-L-rhamnose from UDP-D-glucose (27) or the conversion of polysaccharide-bound beta-D-glucuronic acid to alpha-L-iduronic acid in the course of heparin or dermatan sulfate biosynthesis(28, 29) .

These two classes of sugar epimerization reactions can be distinguished experimentally. The second class of reactions is accompanied by the incorporation of ^3H from ^3H-labeled water into the sugar (29, 30) and by the exchange of [^3H]hydrogen atoms from the epimerization sites of the hexosyl moieties with protons of the medium(31) . When the reaction involves NAD or NADP-bound enzymes, these exchanges of ^3H atoms will not occur.

To distinguish between these two possibilities, slices of tunic were incubated with D-glucose labeled with tritium at C-2, C-3, C-5, or C-6. The [^3H]hydrogen atoms at C-2, C-3, and C-5 are exchanged with protons of the medium and do not remain in the hexosyl moieties after epimerization to L-galactose (Fig. 2, A and B). In contrast, the [^3H]hydrogen atom at C-6 of D-glucose appears in the L-galactose (Fig. 2C). This result shows that the C-6 position is not inverted during the conversion of D-glucose to L-galactose. (^2)


Figure 2: Identification of the ^3H-labeled sugars in the acid hydrolysates. Slices of ascidian tunic (15 mg, wet weight) were incubated with 20 µCi (0.66 nmol) of D-[3-^3H]glucose (panel A), D-[5-^3H]glucose (panel B), and D-[6-^3H]glucose (panel C) for 6 h at 20 °C. The ^3H-labeled compounds were extracted from the tissue slices as described under ``Experimental Procedures'' and in the legend of Fig. 1. The acid hydrolysates of the ^3H-labeled compounds were applied to Whatman No. 1 paper and chromatographed in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h. The chromatograms were cut into 3-mm-wide strips and counted in a liquid scintillation counter. The upper part of the figure shows the chromatographic migration of D-galactose (1), D-glucose (2), and D-mannose (3) standards. Distribution of the ^3H-labeled sugars after labeling the tunic with D-[2-^3H]glucose is similar to that shown in panel A.



These results indicate that conversion of D-glucose to L-galactose involves exchanges of hydrogen atoms at the epimerization sites with protons of the medium.

D-Glucose Is Converted into L-Galactose during the D-[^14C]Glucose Pulse and during the Chase Period

When the ascidian tunic is incubated with D-[U-^14C]glucose, the radioactivity incorporated in the form of D-[^14C]glucose increases rapidly up to 4 h and thereafter remains constant until the end of the labeling period (Fig. 3, A-F; open circles in Fig. 3, G and H). In contrast, the amount of radioactivity that appears as L-[^14C]galactose increases slowly in the first 4 h (closed circles in Fig. 3, G and H).



Figure 3: Identification of the ^14C-labeled sugars in the acid hydrolysates of the molecules from the ascidian tunic labeled for different periods of time. Slices of the ascidian tunic were incubated with D-[U-^14C]glucose for 1 (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 6 (panel E), and 8 h (panel F). The ^14C-labeled molecules were extracted as described under ``Experimental Procedures,'' and their acid hydrolysates were separated and quantitated by paper chromatography as described in the legend of Fig. 1C. The relative amounts (percentage) of each of the ^14C-labeled sugars, on the basis of the area of each peak compared with the total area, are shown beside each peak. Panels G and H show the absolute amounts of label incorporated into each sugar; D-glucose (circle), L-galactose (bullet), and D-mannose (up triangle). The data of panel G were obtained from the experiment shown in panels A-F, and those of panel H were from a similar experiment not shown in the figure.



An explanation for the experiment of Fig. 3is that epimerization of D-glucose to L-galactose occurs after incorporation of D-glucose into the polymer, as described previously for the conversion of polysaccharide-bound beta-D-glucuronic acid to alpha-L-iduronic acid during biosynthesis of heparin and dermatan sulfate(28, 29) . The experiment of Fig. 4supports this possibility. When the D-[^14C]glucose is removed from the incubation medium after 6 h, the content of radioactivity appearing as L-[^14C]galactose increases in the absence of free D-[^14C]glucose (Fig. 4, A-C, D-F, and closed circles in G).



Figure 4: Chase experiment of the ^14C-labeled molecules of the ascidian tunic. Slices of the ascidian tunic were labeled by incubation with D-[U-^14C]glucose for 6 h at 20 °C in two independent experiments (panels A-F), as described in the legend of Fig. 1. The incubation medium was removed, a new medium without D-[^14C]glucose was added, and the tissue slices were incubated for different periods of time, as indicated in panels B-C and E-F. The ^14C-labeled compounds were extracted as described under ``Experimental Procedures.'' Panels A-F, identification of the ^14C-labeled sugars in the acid hydrolysates was performed by paper chromatography, as described in the legend of Fig. 1C. Panel G shows the absolute amounts of label incorporated into L-galactose (bullet) and D-glucose (circle) in Experiment 2 (panels D-F). Panel H, distribution of ^14C between F-1 and F-2 fractions during the chase experiment shown in panels D-F. The ^14C-labeled polysaccharides were analyzed by agarose gel electrophoresis as described in the legend of Fig. 1B. The radioactive bands corresponding to the electrophoretic migrations of standard F-1 (bullet) and F-2 (circle) fractions were scraped into 5 ml of 0.5% PPO/toluene solution and counted in a liquid scintillation counter.



In the tunic of the ascidian S. plicata, the sulfated polysaccharides occur as three main fractions, which are markedly distinct in their molecular weight and chemical composition(2, 3) . The high molecular weight fraction, denominated F-1, contains a high proportion of L-galactose and minor amounts of D-glucose, whereas the other two fractions, of low molecular weight, denominated F-2-A and F-2-B, contain, besides L-galactose, a higher proportion of D-glucose, amino sugars, and small amounts of mannose. Therefore, the pronounced changes in the proportions of L-[^14C]galactose and D-[^14C]glucose during the chase experiment (Fig. 4, A-C, D-F, and G) might reflect modification in the proportions of F-1 and F-2 fractions. However, the experiment of Fig. 4H excludes this possibility. The proportions of ^14C-labeled fractions F-1 and F-2 remain constant during the chase experiment.

Anion Exchange Chromatography on DEAE-cellulose Separates the ^14C-Labeled Molecules of the Ascidian Tunic into Sulfated Polysaccharides and a Nonacidic Compound

The changes observed in the amounts of D-[^14C]glucose and L-[^14C]galactose units during the pulse (Fig. 3) and pulse-chase (Fig. 4) experiments might arise either from modifications in the sugar composition of a single polysaccharide or from interconversion of two types of glycoconjugates. To investigate these possibilities, the ^14C-labeled compounds obtained from pulse and pulse-chase experiments were analyzed by anion exchange chromatography on DEAE-cellulose (Fig. 5, A-C). Two major peaks were observed. One peak was not retained in the column and probably corresponds to a nonacidic compound. The other peak was eluted at high salt concentration and coincides with a peak in metachromaticity (Fig. 5, A-C), which corresponds to the sulfated polysaccharides(2) . Interestingly, the amount of ^14C-labeled nonacidic compound increases during the pulse period (open circles, Fig. 5D) but decreases sharply after the removal of D-[^14C]glucose from the incubation medium. In contrast, the amount of ^14C-labeled sulfated polysaccharides (filled circles, Fig. 5D) increases during both pulse and chase periods.

The distribution of label between D-glucose and L-galactose residues in the nonacidic compound and the sulfated polysaccharides in Fig. 5(A-C) was examined following acid hydrolysis. The nonacidic compound is labeled exclusively in D-[^14C]glucose residues, and the sulfated polysaccharides have a high proportion of L-[^14C]galactose (Fig. 6, A-C), which is similar to the proportion of L-galactose found in the nonlabeled sulfated polysaccharides from the ascidian tunic (Fig. 6E). Therefore, it appears that the ^14C-labeled sulfated polysaccharides obtained during the three experimental conditions of Fig. 5(A-C) are the ``final'' biosynthetic product. In addition, most of the L-[^14C]galactose units of these polymers resist periodate oxidation (Fig. 6D), indicating that these residues are sulfated. In the ascidian polysaccharides, galactose exists mainly as alpha-L-galactopyranosides, linked glycosidically through positions 1-4 and sulfated at position 3 (3, 4, 5, 6, 7) . Thus, the sulfated residues can be distinguished from the nonsulfated units by their resistance to periodate oxidation.


Figure 6: Distribution of ^14C between L-galactose and D-glucose residues in the nonacidic compound and in the sulfated polysaccharides. Panels A-C, the nonacidic compounds and the sulfated polysaccharides from the pulse and chase experiments of Fig. 5, A-C, were subjected to acid hydrolysis (6 N trifluoroacetic acid at 100 °C for 4 h). The mixtures of ^14C-labeled sugars were applied to Whatman No. 1 paper and separated by descending chromatography in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h. The proportions of ^14C-labeled sugars were estimated as described in the legend of Fig. 1C. Panel D, another sample of sulfated polysaccharides labeled for 6 h with D-[U-^14C]glucose, which was submitted to periodate oxidation and borohydride reduction, as described(6) . The proportions of L-[^14C]galactose and D-[^14C]glucose units in this chemically modified polysaccharide were determined as described above. Panel E, nonlabeled sulfated polysaccharide was submitted to acid hydrolysis, and the mixture of sugars was separated by paper chromatography, as described for the ^14C-labeled compounds. The nonlabeled sugars were visualized in the chromatogram by silver nitrate staining and quantitated by densitometry (see (2) for additional details).



Overall, the experiments of Fig. 5and Fig. 6demonstrate that variations in the amounts of D-[^14C]glucose and L-[^14C]galactose units observed during the pulse (Fig. 3) and pulse-chase (Fig. 4) experiments in fact reflect changes in the amounts of ^14C-labeled nonacidic compound and ^14C-labeled sulfated polysaccharides in the ascidian tunic under the different experimental conditions.

The Nonacidic Compound Is Trehalose

The nonacidic compound was extracted from the ascidian tunic and separated from the sulfated L-galactan by anion exchange chromatography on DEAE-cellulose (Fig. 7A). The nonacidic compound has a high specific activity (compare filled and open circles in Fig. 7A). In fact, this compound is almost undetectable by colorimetric assays for hexose. In contrast, the sulfated L-galactan shows a low level of radioactivity per total sugar. Further purification of the nonacidic compound was carried out on a Bio-Gel P-4 column (Fig. 7B), where it eluted as a disaccharide.

The purified ^14C-nonacidic compound migrates as trehalose (71% of the total radioactivity) on paper chromatography (Fig. 8A), releases exclusively D-[^14C]glucose after acid hydrolysis (Fig. 8B), does not migrate on paper electrophoresis at pH 5.0 (Fig. 8C), resists alpha-D-glucosidase (Fig. 8D) and beta-D-glucosidase (Fig. 8E), but is degraded by trehalase (Fig. 8F). Upon the action of this specific enzyme it releases exclusively D-[^14C]glucose. Overall, these experiments demonstrate unequivocally that the nonacidic compound is mainly the disaccharide trehalose. (^3)


Figure 8: Characterization of the nonacidic compound as trehalose. The nonacidic compound (2,000 cpm), previously purified on anion exchange and gel filtration columns (Fig. 7, A and B), was applied to Whatman No. 1 paper, before (panel A) and after incubation with alpha-D-glucosidase (panel D), beta-D-glucosidase (panel E), and trehalase (panel F), and chromatographed in isobutyric acid, 1.0 N NH(4)OH (5:3, v/v) for 24 h. In panel B, the acid hydrolysate (6.0 N trifluoroacetic acid at 100 °C for 4 h) of the ^14C-labeled nonacidic compound was applied to Whatman No. 1 paper and chromatographed in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h. In panel C, the ^14C-nonacidic compound (2,000 cpm) was applied to Whatman 3MM chromatographic paper and submitted to electrophoresis in 0.3 M pyridine/acetate buffer (pH 5.0), run for 4 h at 500 V. The chromatograms and paper electrophoresis were cut into 3-mm-wide strips and counted in a liquid scintillation counter. The upper portion of the panels shows a strip of the chromatograms or of the paper electrophoresis where standard solutions were applied, containing 20 µg each of cellobiose (1), trehalose (2), sucrose (3), D-glucose (4), D-galactose (5), D-mannose (6), D-glucuronic acid (7), and D-glucosamine (8), and the strip was developed with silver nitrate. Incubation conditions are described under ``Experimental Procedures.''



L-Galactose Is Not Detectable among the Intermediates of the Metabolic Route

Attempts to isolate intermediates of the D-glucose sulfated L-galactan metabolic route after incubating homogenates of the ascidian tunic with D-[^14C]glucose were unsuccessful(20) . An alternative attempt to isolate metabolic intermediates involved papain digestion of the tissue slices after pulse and chase with D-[U-^14C]glucose (Fig. 9). The ^14C-labeled polysaccharide + [^14C]trehalose stay at the origin of the chromatograms (Fig. 9, B and C). Products with the same mobility on paper chromatography as standard uridine 5`-diphosphate D-glucose and D-glucose 6-phosphate were identified besides D-[^14C]glucose (Fig. 9E). Two other unknown products were also detected on the chromatograms (X and Y, Fig. 9, B and E). Acid hydrolysis of [^14C]uridine 5`-diphosphate D-glucose, D-[^14C]glucose 6-phosphate, and compounds X and Y forms exclusively D-[^14C]glucose. The amounts of these compounds increase during the 6-h pulse (Fig. 9E) but decrease markedly in the chase phase (Fig. 9F). In contrast, the amounts of ^14C-labeled sulfated L-galactan + [^14C]trehalose increase during the pulse with D-[^14C]glucose and remain constant in the chase period (Fig. 9, A-C).


Figure 9: Attempts to isolate intermediates of the D-glucose sulfated L-galactan metabolic route. Slices of ascidian tunic (15 mg, wet weight) were incubated at 20 °C with 0.7 µCi (1.05 nmol) of D-[U-^14C]glucose in 100 µl of the incubation medium for 1 min (panels A and D) and 6 h (panels B and E). In panels C and F the ascidian tissue was incubated with D-[U-^14C]glucose for 6 h (as in panels B and E), but then the incubation medium was removed, and a new medium without D-[U-^14C]glucose was added to the tissue slices and incubated for additional 3 h. At the end of the incubation periods, the medium was decanted, and the slices were washed 10 times with the incubation medium and then incubated with twice crystallized papain (30 µg) in 100 µl of 0.1 M sodium acetate buffer (pH 5.0), containing 5 mM EDTA and 5 mM cysteine, and incubated at 60 °C for 24 h. The clear supernatants were applied to Whatman No. 1 paper and separated by descending chromatography in isobutyric acid, 1.0 N NH(4)OH (5:3, v/v) for 24 h. The chromatogram was cut transversely into 3-mm-wide strips, which were counted in a liquid scintillation counter. The upper portion of panel D shows a strip of the chromatogram where a standard solution was applied, containing 20 µg each of uridine 5`-diphosphate D-glucose (1), D-glucose 6-phosphate (2), and D-glucose (3); the sugar nucleotide was localized using UV light, and the other sugars were developed with silver nitrate. In panels D-F, distribution of radioactivity in the 5-20-cm regions of the paper chromatograms in panels A-C are shown on an expanded scale (right ordinate).



The absence of detectable amounts of L-galactose among the D-glucose derivatives isolated from tunic slices, together with the unequivocal evidence that this sugar appears in the ascidian polysaccharides, indicates that L-galactose intermediates, if present, are formed in minimal amounts. An alternative explanation is that epimerization occurs after incorporation of D-glucose into the polymer. In fact, galactose exists in the ascidian polysaccharides mainly as alpha-L-galactopyranosides, linked glycosidically through positions 1-4(3, 4, 5, 6, 7) . The small amounts of glucose units occur in these polymers mostly as 4-linked, beta-D-glucopyranosyl residues(3) . Thus, the positions involved in the glycosidic linkage are not inverted during the conversion of these two types of residues (see Fig. Z1).


Figure Z1: Structure 1




CONCLUSIONS AND FUTURE PERSPECTIVES

We investigated the epimerization of D-glucose to L-galactose during biosynthesis of a sulfated L-galactan in the ascidian tunic. Attempts to prepare homogenates from this tissue were unsuccessful since the tunic is rigid and composed of large amounts of fibers and amorphous substance and very few cells(33) . Here we describe a more successful alternative using in vitro pulse labeling of tunic slices. This assay reveals that D-[^14C]glucose is incorporated into the ascidian tunic (Fig. 1A) and epimerizes into L-[^14C]galactose (Fig. 1C) during the biosynthesis of sulfated polysaccharides (Fig. 1B). The interconversion of these two sugars involves exchanges of hydrogen atoms at the epimerization sites with protons from the medium (Fig. 2).

The radioactivity incorporated into molecules of the ascidian tunic as D-[^14C]glucose units increases rapidly up to 4 h and thereafter remains constant until the end of the labeling period (Fig. 3). When the D-[^14C]glucose is removed from the incubation medium after 6 h, the radioactivity incorporated as L-[^14C]galactose increases markedly during the chase period (Fig. 4). These changes in the content of the two sugars in the ascidian tunic in fact reflect changes in the proportions of ^14C-labeled sulfated L-galactan and a ^14C-labeled nonacidic compound (Fig. 5D), which was identified as trehalose (Fig. 8).

Apparently, accumulation of trehalose in the ascidian tissue is a requirement for the synthesis of the sulfated L-galactan from D-glucose (Fig. 3). During the pulse-chase experiments, consumption of trehalose is accompanied by an increase in the synthesis of sulfated L-galactan ( Fig. 4and 5D). In addition, trehalose is synthesized rapidly by cells of the tunic (Fig. 3) but does not accumulate in the tissue (Fig. 7A), suggesting that in ascidians, this disaccharide does not have its usual energy storage function (34) . (^4)

The results obtained in the present investigation permit us to speculate that trehalose may be associated with the biosynthesis of the sulfated L-galactan, perhaps even acting as a donor of the sugar units. Although this proposal is at odds with the many published reports that show sugar diphosphate nucleotides as intermediates in the biosynthesis of glycoconjugates, biosynthetic pathways involving disaccharides are not unknown. A variety of oral streptococci and other microorganisms can utilize sucrose as a donor substrate in the course of dextran synthesis catalyzed by glucosyltransferase(35) . These reactions are possible since the DeltaG of the alpha-linkage of sucrose (and possibly trehalose) is -6.6 kcal/mol, whereas the DeltaG values for the glycosidic linkages range from -2.0 to -4.6 kcal/mol(36) . In this case, conversion of D-glucose to L-galactose would occur after incorporation of D-glucose into the polymer. In fact, attempts to isolate metabolic intermediates involved in the biosynthesis of the sulfated L-galactan reveal exclusively compounds that migrate as uridine 5`-diphosphate D-glucose and D-glucose 6-phosphate, which are precursors of trehalose, and two other compounds containing D-glucose. No intermediate containing L-galactose was identified (Fig. 9).

We attempted to incorporate [^14C]trehalose into the sulfated L-galactan using the in vitro pulse labeling of the tunic slices (as in Fig. 1but with [^14C]trehalose in place of D-[^14C]glucose). No radioactivity was incorporated into the polysaccharides (not shown). This result may be because of difficulties in the transport of trehalose through the cell membrane. We will have to develop a cell-free system, perhaps starting with cells derived from the tunic and grown in culture, to test this hypothesis.


FOOTNOTES

*
This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: FNDCT and PADCT) and Financiadora de Estudos e Projetos (FINEP). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 55-21-270-3443; Fax: 55-21-270-8647.

(^1)
The abbreviation used is: PPO, 2,5-diphenyloxazole.

(^2)
We attempted to incorporate [^3H]H(2)O into the ascidian polysaccharides in vitro using the protocol described under ``Experimental Procedures'' for pulse labeling of these polymers with radioactive sugars. No detectable radioactivity was incorporated. This result may reflect the dilution of [^3H]H(2)O in the tunic slice and/or the low biosynthetic rate of the sulfated L-galactan. Incorporation of ^3H from the medium has been shown in other systems but using cell-free systems and partially purified enzymes(30, 32) .

(^3)
[^14C]Trehalose extracted from the ascidian tunic stays at the origin of paper chromatograms (Fig. 9, B and C). However, after purification on anion exchange and gel filtration columns (Fig. 7), the [^14C]trehalose migrates on paper chromatograms together with the unlabeled standard (Fig. 8A). Our explanation for these results may be that [^14C]trehalose, when first extracted from ascidian tissue by papain digestion, is bound to other molecules, which are removed during the purification procedures.

(^4)
The body of S. plicata (intestine and branchia) does not contain sulfated L-galactan (19) but incorporates D-[^14C]glucose. However, the ^14C-labeled molecules do not migrate in agarose gel electrophoresis, there is no conversion of D-[^14C]glucose to L-[^14C]galactose, and [^14C]trehalose is not formed. Most of the radioactivity is incorporated into a high molecular weight molecule, possibly a glycogen-like polymer. These results reinforce the association between trehalose and the biosynthesis of the sulfated L-galactan in the ascidian tunic, as suggested.


ACKNOWLEDGEMENTS

We are grateful to Adriana A. Eira for technical assistance and to Dr. Martha M. Sorenson for the help in the preparation of this manuscript.


REFERENCES

  1. Albano, R. M., and Mourão, P. A. S. (1983) Biochim. Biophys. Acta 760, 192-196
  2. Albano, R. M., and Mourão, P. A. S. (1986) J. Biol. Chem. 261, 758-765 [Abstract/Free Full Text]
  3. Mourão, P. A. S., and Perlin, A. S. (1987) Eur. J. Biochem. 166, 431-436 [Abstract]
  4. Pavão, M. S. G., Albano, R. M., Lawson, A. M., and Mourão, P. A. S. (1989) J. Biol. Chem. 264, 9972-9979 [Abstract/Free Full Text]
  5. Pavão, M. S. G., Mourão, P. A. S., and Mulloy, B. (1990) Carbohydr. Res. 208, 153-161 [CrossRef][Medline] [Order article via Infotrieve]
  6. Albano, R. M., Pavão, M. S. G., Mourão, P. A. S., and Mulloy, B. (1990) Carbohydr. Res. 208, 163-174 [CrossRef][Medline] [Order article via Infotrieve]
  7. Santos, J. A., Mulloy, B., and Mourão, P. A. S. (1992) Eur. J. Biochem. 204, 669-677 [Abstract]
  8. Mourão, P. A. S., and Bastos, I. G. (1987) Eur. J. Biochem. 166, 639-645 [Abstract]
  9. Vieira, R. P., and Mourão, P. A. S. (1988) J. Biol. Chem. 263, 18176-18183 [Abstract/Free Full Text]
  10. Vieira, R. P., Mulloy, B., and Mourão, P. A. S. (1991) J. Biol. Chem. 266, 13530-13536 [Abstract/Free Full Text]
  11. Vieira, R. P., Pedrosa, C., and Mourão, P. A. S. (1993) Biochemistry 32, 2254-2262 [Medline] [Order article via Infotrieve]
  12. Ribeiro, A. C., Vieira, R. P., Mourão, P. A. S., and Mulloy, B. (1994) Carbohydr. Res. 255, 225-240 [CrossRef][Medline] [Order article via Infotrieve]
  13. Mulloy, B., Ribeiro, A. C., Alves, A. P., Vieira, R. P., and Mourão, P. A. S. (1994) J. Biol. Chem. 269, 22113-22123 [Abstract/Free Full Text]
  14. Pavão, M. S. G., Albano, R. M., and Mourão, P. A. S. (1989) Carbohydr. Res. 189, 374-379 [CrossRef]
  15. Bell, D. J., and Baldwin, E. (1941) J. Chem. Soc. 125-131
  16. Anderson, E., and Lowe, H. J. (1947) J. Biol. Chem. 168, 284-297
  17. Correa, J. B. C., Dmytraczenko, A., and Duarte, J. H. (1967) Carbohydr. Res. 3, 445-452 [CrossRef]
  18. Painter, T. J. (1983) in The Polysaccharides (Aspinall, G. O., ed) vol. 2, pp. 195-285, Academic Press, New York
  19. Pavão, M. S. G., Rodrigues, M. A., and Mourão, P. A. S. (1994) Biochim. Biophys. Acta 1199, 229-237 [Medline] [Order article via Infotrieve]
  20. Mourão, P. A. S. (1991) Biochemistry 30, 3458-3464 [Medline] [Order article via Infotrieve]
  21. Stambuk, B. U., Crowe, J. H., Crowe, L. M., Panek, A. D., and Araujo, P. S. (1993) Anal. Biochem. 212, 150-153 [CrossRef][Medline] [Order article via Infotrieve]
  22. Farndale, R. W., Buttle, D. J., and Barret, A. J. (1986) Biochim. Biophys. Acta 883, 173-177 [Medline] [Order article via Infotrieve]
  23. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350-356
  24. Avigad, G., Amaral, D., Ascencio, C., and Horecker, B. L. (1962) J. Biol. Chem. 237, 2736-2743 [Free Full Text]
  25. Adams, E. (1976) Adv. Enzymol. 44, 69-138 [Medline] [Order article via Infotrieve]
  26. Slein, M. W. (1955) Methods Enzymol. 1, 299-306
  27. Gabriel, O. (1978) Trends Biochem. Sci. 3, 193-195
  28. Malmstrom, A., Rodén, L., Feingold, D. S., Jacobsson, I., Backstrom, G., and Lindahl, U. (1980) J. Biol. Chem. 255, 3878-3883 [Abstract/Free Full Text]
  29. Prihar, H. S., Campbell, P., Feingold, D. S., Jacobsson, I., Jensen, J. W., Lindahl, U., and Rodén, L. (1980) Biochemistry 19, 495-500 [Medline] [Order article via Infotrieve]
  30. Barber, G. A. (1980) J. Biol. Chem. 254, 7600-7603 [Abstract]
  31. Jacobsson, I., Backstrom, G., Hook, M., Lindahl, U., Feingold, D. S., Malmstrom, A., and Rodén, L. (1979) J. Biol. Chem. 254, 2975-2982 [Medline] [Order article via Infotrieve]
  32. Feingold, D. S. (1982) Encycl. Plant Physiol. New Ser. 13A, 37-39
  33. Barnes, R. D. (1980) Invertebrate Zoology , pp. 1030-1032, W. B. Saunders Co., Philadelphia
  34. Panek, A. D. (1991) in The Yeasts (Ross, A. H., and Harrison, J. S., eds) Vol. 4, pp. 655-678, Academic Press, New York
  35. Mayer, R. M. (1987) Methods Enzymol. 138, 648-661
  36. Leloir, L. F., Cardini, C. E., and Cabib, E. (1960) Comp. Biochem. 2, 117

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.