Some structural studies on the galactose-containing polysaccharide from bovine placenta

Placenta(1993), 14, 439-448

Some Structural Studies on the GalactoseContaining Polysaccharide from Bovine Placenta R. P O N T A R O L O , M . A. L A C O M B E

a'c J. H . D U A R T E b & FEIJ6 b

aDepartamentos de Farmdcia e de bBioquimica, Universidade FederaldoParand, CaixaPostal, 19046, 81.531 Curitiba, Parand, Brazil. c To whom correspondenceshould be addressed Paper accepted6.10.1992

SUMMARY Polysaccharides were extractedfrom 8-month-old placenta with aqueous HgCl2. The protein-free material was purified by selective precipitation with Cetavlon in the presence of sodium borate at p H 8.5 and was homogeneous on molecular-sieve chromatography, electrophoresis, and on treatment with Concanavalin A. The preparation contained galactose and glucose as principal monosaccharides with 5per cent of hexosamines. Methylation studies suggested that 9-gluco and D-galactopyranosyl units may be constituents of glucan and galactan respectively which form a molecular aggregate that does not dissociate during the fractionation procedures. After treatment of thefraction with fl-amylase, the proportion ofglucose in the polysaccharide diminished, indicating the presence of (1---~4)-linked a-Dglucopyranosyl residues. Also, when the fraction was treated with a crude protease having glucosidase activity a residual a-D-galactopyranan was isolated and found to contain non-reducing end-groups (30. Oper cenO, 3 - 0 - (39.5per cent) and 3,6-diO-substituted (30.5per cent) units. The structure of the galactan was not modified according to methylation data, on removal of the glucosyl component. The polysaccharide fraction (pH 8.5 Cetavlon), isolated from bovine placenta, thus contains a glycogen-like material associated with a galactan as molecular aggregate. This galactan has not been previously recognized in bovine placenta and its occurrence in this organ supports the hypothesis that galactose-containing polysaccharides are involved in foetal development.

INTRODUCTION The occurrence of glycogen in placental tissue was first evidenced in rabbit placenta by Claude Bernard in 1859. He suggested that glycogen in this organ could s e r v e a s a r e s e r v e carbohydrate source for the fetus until it acquired the capacity of synthesizing it. 0143-4004/93/040439 + 10 $08.00/0

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Several other references to the presence of glycogen in the placenta of other species, occur in the literature namely humans (Corey, 1985; Driessen, 1907; Villee, 1976), guinea-pigs (Yarimagan and Bor, 1981), rats (Villee, 1976) and rabbits (Bernard, 1859; Huggett and Morrison, 1955). The presence of glycogen in human placenta was first demonstrated by Drissen in 1907 using histological techniques, and some of its structural aspects were investigated (Blows et al, 1988). The glycogen content in placenta varies according to the gestation time. In human placenta, the polysaccharide content is high at the beginning of pregnancy, decreasing with its advance (ViUee, 1976). On the other hand, rat and rabbit placenta have a low glycogen content at the beginning of pregnancy increasing to a maximum at two-thirds of the gestation period, thereafter decreasing (Huggett and Morrison, 1955). Lacombe Feij6 and Vieira Lopes (1984) studied bovine placenta after 4 months of pregnancy and obtained a polysaccharide fraction having glucose (80 per cent) and galactose (20 per cent). Pontarolo et al (1990) investigated the D-glucose to D-galactose ratio in the bovine placenta as a function of gestation time, and showed that there is little variation from the third to the sixth month. However, a significant decrease in the percentage of D-glucose was observed from the sixth to the ninth month (68 to 45 moles per cent) with a corresponding increase in D-galactose. The lack of change of the D-glucose to D-galactose ratio from the third to the sixth month can be attributed to the dynamic equilibrium between the polymers having different molecular structures with the degradation and synthesis velocities tending to assume constant values. Although numerous investigations related to the carbohydrate metabolism in the embryonic and fetal period in various animals have been carried out, there is no mention of the presence of galactan in placenta tissue. This work has aimed at investigating some structural aspects of an isolated polysaccharide fraction of bovine placenta between the eighth and ninth month of pregnancy.

MATERIALS AND M E T H O D S General methods Optical rotations were measured in water with a Perkin Elmer model 241 polarimeter at 25~ Electrophoresis on cellulose acetate strips (Cellogel) was carried out with samples dyed with Procion Blue, using 0.05 M sodium tetraborate buffer (pH 9.2) according to the method of Dudman and Bishop (1968). GLC analysis was carried out with a Varian Gas Chromatograph Model 2440 (flame ionization detector), column (120 x 0.2 cm i.d.) packed with 3 per cent ECNSS-M (w/w) on 100-200 mesh Gas Chrom Q a t 160~ (Gorin et al, 1979) with nitrogen as carrier gas at 37.5 ml/min. The triangulation procedure was used in quantitative analyses (Sawardeker, Sloneker and Jeanes, 1965). 13C-NMR spectra were obtained with a AM-360W Bruker Spectrometer incorporating Fourier Transform using D20 as solvent at 33~ Employed spectrum parameters were spectral width 20 000 Hz, acquisition time 0.10 s, pulse width 21.0 ~ts, and number of transients 11 792. Chemical shifts were expressed in 6 (ppm) relative to the tetramethylsilane resonance (6 = 0), determined in a separate experiment. Paper chromatograms were obtained using Whatman No. 1 filter paper with one of the

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441

o-a tt3

cO

e4

cO tt'~t',. 00 Lea

Cq

Figure 1. 13C-NMRspectrumofFP1 for a solutionofdeuteriumoxideat 33~ Depictedvaluesare chemicalshifts

expressedin 6(ppm)relativeto an external standard oftetramethylsilane.

following systems (v/v): (a) benzene/butan-l-ol/pyridine/water (1:5:3:3), (b) butanone/ water/ammonium hydroxide (200:17:1). Sugars were detected with alkaline silver nitrate (Trevelyan, Procter & Harrison, 1950), p-anisidine hydrochloride (Hough, Jones and Wadman, 1950), and ninhydrin reagents. Sugar mobilities on paper chromatograms are expressed as RGAL,compared with that ofD-galactose (RGAL).Quantitative determination of total protein and sugars was carried out respectively by the Lowry et al (1951) and phenolsulphuric acid methods (Dubois et al, 1965). Sulphate groups were determined by the process described by Antonopoulos (1962). Absorption spectra of glycogens were determined in the presence of iodine-iodide and saturated calcium chloride as described by Krisman (1962). Isolation and purification of placental polysaccharide fraction. Placenta were obtained from the

Argus slaughter house (in S~o Joss dos Pinhais, PR) from cows in their eighth and ninth month of pregnancy. The pregnancy period was determined taking into consideration the external morphological characteristics of foetus and also by the method of Hertzel (Mies Filho, 1975). Placenta were removed immediately after slaughter and then maintained at 0~ Placental nodules (constituted ofcaruncula and fetal cotyledon) free from uterine tissue and from corium-alantoide membranes, were weighed (8.5 kg), homogenized (Waring blender), and deproteinized with 3 per cent aqueous HgClz having 1 ml/tissue gram as described by Pontarolo et al (1990). The acetone powder (22.8 g; ca 1.5 per cent of the dry weight) was then dissolved in water Cetavlon at pH 7.0 and then in the presence of sodium borate at pH 8.5, as described by Duarte and Jones (1971). The precipitate obtained in pH 7.0 containing polymers including DNA, RNA and glicosaminoglycans was discarded, and that obtained at pH 8.5 was isolated by centrifugation, solubilized in 2 M acetic acid, and precipitated with 3 volumes of ethanol. The resulting precipitate was resuspended in water, dialysed for 72 h and freeze dried, and the fraction called FP1 2.0 g; 8.7 per cent yield, [a]~ + 114 ~ (c, 0.5 in water).

Placenta(1993), Vol. 14

442 e,D

eo

eq

L__A Figure2. lsC-NMRspectrumofFP2 for a solutionofdeuteriumoxideat 33~ Depictedvaluesare chemicalshifts expressedin 6(ppm)relativeto an externalstandard oftetramethylsilane. Gelfiltration. The polysaccharide fraction FP1 (5 mg/ml in water) was applied to a Sepharose column CL-2B (50 • 1.8 cm) which was eluted with a supporting electrolyte (0.2 M NaC1). The eluant was collected in 4 ml fractions, and total sugar and protein contents measured. The void volume (Vo) of the column, was determined by elution with high mol. wt., Dextran blue. Acid hydrolysis offraction FP1. The polysaccharide fraction (FP 1) (20 rag) was hydrolysed with 2 M TFA (4 ml) at 100~ for 15 h. The hydrolysate was examined by paper chromatography, which showed the presence of glucose (52.5 per cent) and galactose (47.2 per cent) as the main constituents. Reduction and acetylation ofhydrolysates. Each hydrolysed sample was reduced with NaBH4 at room temperature for 6 h (pH 9.0). The reaction was interrupted with 2 M acetic acid, sodium ions removed with Amberlite 1R-120 (H+), and boric acid removed by co-distillation with methanol. The product was dried in vacuo, and acetylated with acetic anhydride pyridine (1:1, v/v) for 5 h at 100~ as described by Wolfrom and Thompson (1963). Enzymatic determination of D-glucose in FP1. The acid hydrolysate (TFA) (5 mg) from FP 1 was incubated at 37~ for 1 h with glucose oxidase, peroxidase, and dianisidine, according to the technique described by Dahlqvist (1961); 51.0 per cent of the total sugar, previously estimated by the phenol-sulphuric acid method, was oxidized by the enzyme. Enzymatic determination ofo-galactose. Acid hydrolysed FP1 (5 rag), having 30/~g of reducing sugar (0.4 ml), was incubated with 200 #g of galactose oxidase and 1.6 ml peroxidase o-dianisidine (chromogenic reagent) by the technique described by Amaral, Kelly and Horecker (1966). After 60 min at 37~ 46.5 per cent of the polysaccharide fraction (FP1) was transformed into the dialdehyde. Determination ofhexosamines. The presence of hexosamine was investigated in a hydrolysate obtained from polysaccharide (10 mg) after acid hydrolysis (6 M HC1, 100~ 6 h) according

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tO the process described by Boas (1953). The fraction contained 5 per cent ofhexosamine, characterized as glucosamine and galactosamine by paper chromatography (72 h). Fractionation of FP1 on a DEAE-cdlulose column. DEAE-cellulose column (2.5 x 26 cm) in

chloride, hydroxide and borate forms were prepared as described by Gardell (1965), Neukom and Kuendig (1965). A polysaccharide fraction (20 mg) in water (2 ml) was chromatographed on each of the columns. The samples applied to the column in the chloride form were successively eluted with water, 0.1 M and 0.25 M KC1. The fraction eluted with 0.1 M KC1 corresponded to 70 per cent of the sample applied. All fractions showed the same monosaccharide composition. A sample applied to the column in the hydroxide form was successively eluted with water, 0.01 M, 0.05 M and 0.12 M NaOH, 90 per cent of the sample being eluted with 0.01 M NaOH. No elution was observed with water. A sample applied to column in the borate form was eluted stepwise with water, 0.01 M and 0.25 M NazB407, water at pH 7.0 and finally with acetic acid 0.5 M and 1.0 M. No polysaccharide was eluted with water. Three fractions were obtained by elution with aqueous NazB407 (24, 17, 12 per cent respectively). There were no significant qualitative differences in the monosaccharide ratio ofpolysaccharide eluted in each fraction. Methylation ofFP1. FP1 (100 mg) was methylated four times by the Haworth procedure

(1915). The reaction mixture was neutralized (dilute sulphuric acid) and salts removed by dialysis against water and evaporated to dryness. Methylation was completed using the Hakomori (1964) procedure, as modified by Sandford and Conrad (1966). The solution was neutralized (3 M sulphuric acid), extracted with chloroform and concentrated (yield 50 per cent). The resulting methylated polysaccharide showed no infrared absorption for hydroxyl groups at 3400 cm -1. Methanolysis of methylated FP1. Methylated FP1 (50 mg) was treated with 5 per cent

methanolic hydrogen chloride (10 ml) for 5 h at 100~ as described by Bouveng and Lindberg (1965). The cooled solution was neutralized with silver carbonate. The mixture of resulting methyl glycosides was hydrolysed with 0.5 M HC1 (5 ml) for 5 h at 100~ as described by Parikh and Jones (1966). The cooled solution was filtered and lyophilized. The methylated reducing sugars were treated with sodium borohydride and the products acetylated (acetic anhydride/pyridine). The resulting partially methylated alditol acetates were analysed by GLC (Table 1). Table 1. GLC analysisofpartiallyO-methylatedald~tolacetatesobtainedfromFP1

and FP2 Moles % formedfrom PartiallyO-methylatedalditolacetate*

T**

FP1

FP2

2,3,4,6-Me4-Gluc 2,3,6-Me3-Gluc 2,3-Mez-Gluc 2,3,4,6-Me4-Gal 2,4,6-Me3-Gal 2,4-Mez-Gal

1.00 2.50 5.60 1.25 2.30 6.60

7.20 85.50 7.30 29.10 42.20 28.70

30.00 39.50 30.50

*Analyseswith colkumnof ECNSS-M at 3 per cent w/w in Gas Chrom Q, at 160012. **Retentiontime(T) relativeto thatof 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-Dglucitol.

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Placenta (1993), Vol. 14 0.3

'~

0.2

o

.~ 0.1

0.00

i

20

I

40

60

80 100 120 Elution volume (ml)

140

160

Figure 3. Elution diagram of FP1 and FP2 on sepharose CL-2B, determined by phenol-sulphuric acid (a reagent). Bed dimensions 1.8 x 50 cm. Element; 0.2 M NaCL Flow rate; 0.4 ml/min. Void volume; 44 ml. e, FP1; n , FP2.

Treatment of FP1 with t-amylase. FP1 was submitted to enzymatic depolymerization with /~-amylase, (1--~4)-a-D-glucan maltohydrolase, of Aspergillus niger (Sigma) according to Manners and Wright (1962). In a solution having 0.4 per cent polysaccharide, 0.5 mM glutathione, 0.05 M serum albumin, 30 mM acetate buffer pH 4.8, and 0.75 ml enzymatic solution (1.0 mg/ml), which was incubated at room temperature (20-22~ in a toluene atmosphere. After 12 h more enzyme solution was added. Incubation was interrupted when the liberated reducing sugar remained constant. The system was spun, and the supernatant dialysed for 24 h. Thereafter, the solution was treated with ethanol (3 volumes), and the resulting precipitate was centrifuged, dissolved in water and lyophilized. A sample was hydrolysed, the hydrolysate reduced with NaBH4 and then acetylated. The alditol acetates obtained were analysed by GLC and characterized as hexaacetates of galacitol (80 per cent) and glucitol (20 per cent). Treatment of FP1 with a enzyme preparation having protease and glucosidase activities. An FP 1 sample (100 mg) was submitted to enzymatic action (A. oryzae~rotease - type II of Sigma, 2 x 10 mg) in 0.005 M CaC12, buffered with ammonium acetate (pH 8.5; 80 ml) for 168 h at 37~ in a toluene atmosphere. The solution was then centrifuged, the supernatant treated with ethanol (3 volumes) and re-centrifuged. The supernatant thus obtained was evaporated to dryness and examined by paper chromatography, which showed the presence of glucose only. The above precipitate was dissolved in water, dialysed for 24 h and then submitted to deproteinization three times by the Sevag method (Staub, 1965). The aqueous fraction obtained after Sevag treatment was lyophilized (55 mg) and called FP2, which was submitted to a Sepharose CL-2B as described, and showed only one symmetric peak (Figure 3). A sample of FP2 (5 mg) was totally acid hydrolysed and paper chromatography showed only galactose and traces ofhexosamines. After NaBH4 reduction and acetylation, GLC analysis showed only the presence of galactitol hexaacetate.

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Periodate oxidation of the polymer FP2. A sample of FP2 (30 mg) was oxidized with 0.01N sodium metaperiodate (100 ml) in the dark for 94 h at 0-2~ Aliquots (5 ml) of the solution were analysed for periodate uptake (Hay, Lewis and Smith, 1965) and formic acid production (Baker and Somers, 1967). The respective values were 0.60 mole/mole and 0.30 mole/mole of anhydrohexose. Smith degradation ofFP2. FP2 (20 mg) was oxidized with sodium m-periodate as described by Hay, Lewis and Smith (1965). The oxidized polysaccharide was reduced with NaBH4, and the polyalcohol hydrolysed with 2 M trifluoroacetic acid (15 h, 100~ Reduction with NaBH4, followed by acetylation, gave a mixture of acetates of glycerol (33.8 mole per cent) and galactitol (66.2 mole per cent) when analysed by GLC with the conventional ECNSS-M column described earlier. Methylation analysis ofFP2. FP2 (20 mg) was methylated four times by the Haworth process and once by the Hakamori procedure. The methylated polysaccharide (15 mg) was treated with 5% methanolic hydrogen chloride (1.5 ml) for 5 h at 100~ The methyl glycosides obtained were treated with 0.5M HC1 (1 ml) for 5 h at 100~ and the product reduced with NaBH4, acetylated and analysed by GLC (Table 1).

RESULTS AND DISCUSSION The polysaccharide fraction FL1 was isolated from the bovine placental nodules (constituted of fetal cotyledon an caruncula) by deproteination with HgCI2, followed by purification with Cetavlon in the presence of borate buffer (pH 8.5). The precipitated fraction FP1 had 4 per cent (w/w) of protein and showed only one band when its polysaccharide portion dyed with Procion Blue was submitted to electrophoresis. Although methylation analysis data suggested the presence of at least two polysaccharide components, FP1 was homogeneous when submitted to chromatography on column ofSepharose CL-2B (Figure 3), with a supporting electrolyte (0.2 M NaC1), with yields of 95 per cent and protein contents of 4 per cent. In Sepharose 6-B and 4B-200, FP1 was eluted in the void volume (Vo). An attempt was also made to fractionate FP1 on columns of DEAE-cellulose (chloride, hydroxide, and borate forms). However, the fractions obtained were not significantly different quantitatively. Acid hydrolysis of FP 1 furnished its main components glucose and galactose, along with a small proportion of hexosamines (ca 5 per cent), characterized as glucosamine and galactosamine by paper chromatography. Sulphate groups were absent. Enzymatic examination using D-glucose oxidase and D-galactose oxidase showed only D-forms to be present. The partially O-methylated alditol acetates formed on methylation analysis of FP1 were examined by GLC. (Table 1). It can be observed that the proportions of non-reducing endunits of glucose correspond to that of glucose branch points. The same is true of those of galactose. The values are 2,3-Mez-Gluc (7.3 per cent), 2,3,4,6-Me4-Gluc (7.2 per cent), and 2,3,6-Me3-Gluc (85.5 per cent) and for the galactan are 2,4-Me2-Gal (28.7 per cent), 2,3,4,6-Me4-Gal (29.1 per cent), and 2,4,6-Me3-Gal (42.2 per cent). This is consistent with FP1 containing a mixture of a branched glucan and a branched galactan. FP1 contained glucose and galactose in a 53:47 molar ratio. After enzymatic treatment with t-amylase this ratio was 80:20. Such a decrease of D-glucose indicates that (1--~4)linked a-D-glucopyranosyl units were present, consistent with the methylation data

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Placenta (1993), VoL 14

(Table 1). The quantity of D-glucose remaining after enzymatic action is compatible with the dextrin limit of a glycogen. The specific rotation of FP1 of + 114 ~is also indicative of a high proportion of a-D-hexopyranosyl units. Experiments carried out with liver glycogen and FP1 show that the former had a )].max 460 nm (with A = 1.011). FP1 showed '~max 400 nm (with A = 0.892). The higher absorption of liver glycogen in the presence of iodine at 460 nm rather than at 520 nm indicates that the liver glycogen had a normal branched structure. The absorption in 400 nm agrees with the presence of limit dextrins in FP1. The polymeric product FP2 remaining after enzymatic reaction would be expected to have a higher proportion ofD-galactose. The specific rotation of D-galactan + 8~is consistent with a fl-D-galactopyranan. As can be observed the structure of the galactose polymer did not vary significantly after enzymatic removal of D-glucose units from FP1 (Table 1). This observation and the absence of methylated D-glucose derivatives in the hydrolysis products of FP2 is in accordance with the suggested presence of two macromolecules in the FP1 fraction (Fig. 3). 13C-NMR signals (Figure 1) from fraction FP1 are in accord with the methylation and specific rotation data. Assignable signals are those of C-1 of fl-D-galactopyranosyl units (6 103.53), C-1 of a-D-glucopyranosyl units (6 100.54) and C1 (6 96.20), C-2 (6 55.69), C = O (6 175.46) and CH3 (6 23.08) of 2-acetamido-2-deoxy-a-D-hexopyranosyl units (Gorin, 1981a; Gorin, 1982b). Small high fields signals arose from protein (4 per cent). The specific rotation ( + 114~ agreed with that of a mixture offl-D-Galp units (+ 22.0~ (Iacomini et al, 1981) and glycogen (+ 197.8~ (Bell and Hosterlits, 1935). After treatment with the enzyme mixture having glucosidase and protease activity, the resulting fraction, FP2, gave a spectrum (Figure 2) lacking the C- 1 signal of glycogen and those of protein. In agreement with the absence of glycogen the specific rotation (+ 8~ was greatly reduced. The C-1 signal at 6 101.50 was not assigned, but on treatment of FP2 with 20 per cent aqueous potassium hydroxide at 100~ followed by precipitation of resulting polysaccharide with ethanol, the signal was not present. De-N-acetylation also occurred as the CH3 and C = O signals were absent. Thus it seems possible that the galactan has a hexosamine component. Consumption ofperiodate and formation of formic acid are also in accordance with the methylation data of FP2 (Table 1) that showed the presence of 30.0 per cent of D-galactopyranosyl units in non-reducing end-groups; 39.5 per cent of 3.O-substituted D-galactopyranosyl units; and 30.5 per cent of 3,6-di-O-substituted D-galactopyranosyl units. Results obtained by Smith degradation indicate the possibility that FP2 consists of 66.2 per cent units not susceptible to oxidation with periodate and of 33.8 per cent units that give rise to glycerol, attributed in this case to the presence of linear chains constituted by galactopyranosyl units, linked by glycosidic bonds (1--~6) type and/or non-reducing terminal units. This proportion is compatible with the periodate consumption data (0.6 mol/mol of anhydro hexose), and formic acid liberated (0.3 mol/mol of anhydro hexose), of the polysaccharide, since the percentage of units that liberate formic acid (30.0 per cent) during oxidation corresponds to the number of units producing glycerol (33.8 per cent) in the terminal group analysis. The possibility of4-O-substituted galactopyranosyl units is not valid because of the absence of the threitol tetraacetate on Smith degradation. The results indicate the possibility that two polysaccharides are present in FP1, a glucan similar to glycogen, and a galactan associated by a molecular aggregation that was indissociable under our conditions. However, the experimental data do not exclude two possibilities that will be investigated in the future. First, the two macromolecules could be bound by a protein bridge. Second, the galactan

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could be bound to the glucan via a small number of glycosidic linkages that would not be

detected by methylation analyses obtained before and after treatment with the protease with glucosidase activity. Thus, the branched galactopyranari, which has not been previously found in bovine placenta, is similar to that already found during the reproductive process of other animals (Iacomini et al, 1981; Lacombe Feij6 and Duarte, 1975), but its real biological role in this organ remains to be elucidated. ACKNOWLEDGEMENTS The authors wish to thank Dr P. A.J. Gorin for his interest and advise, Mr. M. Mazurek (National Research Council of Canada, Plant Biotecnology Institute, Saskatoon, Saskatchewan S7N 0W9, Canada), and Dr A. Zanata (Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil) for running 13C-NMR spectra.

REFERENCES

Amaral, D., Kelly, F. F. & Horecker, B. L. (1996) Galactose oxidase ofPolyporuscircinatus.Methods in Enzymology, 2, 87-92. Antonopoulos, C. A. (1962) A modification for the determination of sulphates in mucopolysaccharides by the benzidine method.Acta ChemicaScandinavica, 16, 1521-1522. Baker, S. A. & Somers, P. J. (1967) A spectrophotometric method for the determination of formic acid on the periodate oxidation of carbohydrates. CarbohydrateResearch, 3, 220-224. Bell, D.J. & Hosterlits, H. (1935) Liver glicogen. II. Acyl derivatives and 'regenerated glycogens'. The Biochemical Journal, 29, 2027. Bernard, C. (1859) De la maturd glicog~ne considerde comme condition de developpement certains tissus chez de le foetus avan't l'a parition de la fonction glicogdnique du foie. Journal PhysiologyHommeAnimaux, 2, 236. Blows,J. M. H., Caz Der, P. C., Geddes, R. & Wills, P. R. (1988) The structure of placental glycogen. Placenta, 9, 493-500. Boas, N. F. (1953) Method for the determination ofhexosamines in tissues.Journal ofBiological Chemistry, 24, 553563. Bouveng, H. & Lindberg, B. (1965) Hydrolysis of methylated polysaccharides. Methods in CarbohydrateChemistry, 5, 296-297. Corey, E. L. (1985) Growth and glycogen content of the fetal liver and placenta.American Journal ofPhysiology, 112, 263-267. Dahlqvist, A. (1961) A determination of maltose and isomaltose activities with a glucose oxidase reagent. BiochemicalJournal, 80, 547-555. Driessen, L. F. (1907) Veber glycogen in der placenta. Achives Gynaek, 82, 278. Duarte,J. H. &Jones,J. K. N. (1971) Some structural studies on the galactan from the albumen glands of the snail Strophocheilus oblongus. Carbohydrateresearch, 16, 327-335. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1965) Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350-356. Dudman, W. F. & Bishop, C. T. (1968) Electrophoresis of dyed polysaccharides on cellulose acetate. Canadian Journal of Chemistry, 46, 3079-3084. Gardell, S. (1965) Fractionation on cellulose column. Methods in CarbohydrateChemistry, 5, 9-14. Gorin, P. A.J., Previato, J. O., Previato, M. L. & Travassos, L. R. (1979) Structure of the D-mannan and Dgalactan in Crithidiafasciculata: change in proportion with age of culture. Journal of Protozoology, 26, 473--478. Gorin, P. A. J. (1981a) Carbon-13 nuclear magnetic resonance spectroscopy of polysaccharides. Advances in Carbohydrate Chemistryand Biochemistry, 38, 13-104. Gorin, P. A. J. (1982b) Preparation of a and fl anomers of various isomeric methyl O-D-galactopyranosyl-Dgalactopyranosides. Standards for interpretation of 13CNMR spectro of D-galactopiranans. CarbohydrateResearch, 101, 13-20. Hakomori, S. (1964) A rapid permethylation of glycolipid and polysaccharide catalyzes by methylsulfinyl carbanion in dimethyl sulfoxide. Journal of Biochemistry, 55,205-208. Haworth, W. N. (1915 ) A new method of preparing alkylated sugars. Journal of the ChemicalSociety, 107, 8-16. Hay, G. W., Lewis, B. A. & Smith, F. (1965) Periodate oxidation ofpolysaccharides: general procedures. Methods in CarbohydrateChemistry, 5, 357-361. Hough, L.,Jones, J. K. N. & Wadman, W. H. (1950) Quantitative analysis of mixtures of sugars by the method of partition chromatography. Part V. Improved method for separation and detection of the sugars and their methylated derivatives on the paper chromatogram.Journal of the ChemicalSodety, 1702-1706.

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Huggett, A. S. G. & Morrison, S. D. (1955) Placental glycogen in the rabbit. Journal of Physiology, 129, 68. laeomini, M., Duarte, G. R., Duarte, H. S., Fontana, J. D. & Duarte, J. H. (1981) Structural study on snail galactans from the genus Biomphalaria.Agricultural and Biological Chemistry. 45, 1373-1380. Krisman, C. R. (1962) A method for the colorimetric estimation of glycogen with iodine.AnalyticalBiochemistry,4, 17-23. Laeombe Feij6, M. A. & Duarte,J. H. (1975) Some structural studies on the focogalactan from egg masses of the snail A mpullarius sp. CarbohydrateResearch, 44, 241-249. Lacombe Feij6, M. A. & Vieira Lopes, L. C. (1984) Galactose containing polysaccharides from placenta and fetal liver during pregnancy in bovine.Arquivos de Biologia e Tecnologia, 27, 9-12. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. V. (1951) Protein measurement with the Folin phenol reagent. Journal ofBiological Chemistry, 193, 265-275. Manners, D. J. & Wright, A. (1962) a-l,4-Glucosans. Part XIII. Determination of the average chain length of glycogens by fl-amylosis.Journal of the ChemicalSoday, 2, 1597-1962. Mies Filho, A. (1975) Reprodu~o dos animais e inseminaq~o artificial. Organiza~o Sulina de Representaqfes. Porto Megre, 213-215. Neukom, H. & Kuendig, W. (1965) Fractionation on diethylaminoethyl-cellulose columns. Methods in Carbohydrate Chemistry, 5, 14-17. Parikh, V. M. &Jones, J. K. N. (1966) Cholla gum. I. Structure of the degraded cholla gum. CanadianJournal of Chemistry, 44, 327-330. Pontarolo, R., Vieira Lopes, L. C., Duarte,J. H. & Laeombe Feij6, M. A. (1990) Studies on the polysaccharide from bovine placenta at different stages of pregnancy.Arquivos de Biologia e Teenologia,33, 226-239. Sandford, P. A. & Conrad, H. E. (1966) The structure oftheAerobacteraerogenes A3 (S1) Polysaccharide. I. A reexamination using improved procedures for methylation analysis. Biochemistry, 5, 1508-1517. Sawardeker, J. S., Sloneker, J. H. & Jeanes, A. (1965) Quantitative determination of monosaccharide as their alditol acetates by gas liquid chromatography. Analytical Biochemistry, 37, 1602-1604. Staub, M. A. (1965) Removal of proteins. Sevag method. Methods in CarbohydrateChemistry, 5, 5-6. Trevelyan, W. E., Procter, D. P. & Harrison, J. S. (1950) Detection of sugars on paper chromatogram by use of dipping reagents. Nature, 166, 444-455. Villee, C. A. (1976) Biochemical aspects of the mammalian placenta. In The Biochemistry of Animal Development. (Ed.) Top, T. New York, Academic Press, 385--412. Wolfrom, M. L. & Thompson, A. (1963) Acetylation. Methods in CarbohydrateChemistry, 2, 282-292. Yarimagan, H. S. & Bor, N. M. (1981) Effects of epinephrine on guinea pig placental glycogen metabolism and on cellular cyclic AMP. BiochemicalMedicine, 25, 125-292.