Ganglioside column

Experiment was carried out as described in Figure 1, using 20 lU/mL of both interferons. , Mouse fibroblast interferon , T-cell interferon , T-cell interferon after passage through a Sepharose—ganglioside column (see Figure 8). T EMC titer in the absence of interferon (19). 

Figure 8. Lack of binding of mouse T-cell interferon to Sepharose-ganglioside columns...

Bio-Sil A was activated at 110°C overnight, suspended in chloroform and packed into a column (1.5 x 45 cm). Fraction I (eluted from DEAE-Sephadex with 0.01 M sodium acetate was dissolved in C M (2 1, v/v) and applied to the column. Gangliosides were eluted with a C M H20 solvent system of increasing polarity. We have been using the following mixtures ... 

Fractions of 6 ml volume were collected and 50 yl aliquots used to identify the gangliosides by TLC. Four gangliosides have been purified from fraction I of chicken skeletal muscle directly from the column. 

Comparison of glycosphingolipids from human and chicken skeletal muscle. The elution of gangliosides from DEAE-Sephadex A 50 column with these 0.01, 0.02 and 0.2 M sodium acetate concentrations separated the gangliosides into mono-, di- and poly-sialo- fractions. The gangliosides of human muscle are shown in Fig. 1A. The monosialogangliosides GM3, GM2 and GM1 were eluted with 0.01 M sodium acetate in methanol (lane 2), GD3 and GDla with the 0.02 M solvent (lane 4) and others with 0.2 M acetate... 

Figure 1A. Thin-layer chromatogram of human and chicken muscle ganglioside fractions eluted from DEAE-A 50 column...

Buffered Tetrahydrofuran. In 1973, Tettamanti et al. [19) described an improved procedure for the extraction, separation and purification of brain gangliosides. In this method, the brain tissue was subjected to homogenization and extraction with buffered [potassium phosphate buffer, pH 6.8) tetrahydrofuran. Following centrifugation, diethyl ether was added and the mixture separated into organic and aqueous phase. The gangliosides, recovered exclusively in the aqueous phase, were then freed of residual phospholipids and other minor contaminants [i.e. peptides)by column chromatography on silica gel.This procedure, as shown by the authors,was superior to the commonly used chloroform/methanol... 

Figure 11. Effect of incubation time (at 37°C) and of ganglioside concentration on the incorporation of gangliosides (G.y, G m, GTib) into phosphatidylcholine mono-lamellar vesicles. Phosphatidylcholine (as vesicles) 9 nmol. Ganglioside from 0.5 to 2 nmol. After incubation the mixtures were passed through a 1 X 20 cm Sepharose 4B column to separate vesicles from ganglioside micelles.

Glycolipid analysis. Gangliosides were extracted with chloroform-methanol (2 1), purified on Sephadex G-25 columns and separated into individual species by thin-layer chromatography as described previously (12). 

Column Chromatography. Sepharose beads containing covalently linked gangliosides (0.2 ml packed volume) were placed into a pasteur pipette containing a small amount of glass wool. Columns were washed with HEM containing 50 ug/ml bovine serum albumin (3 ml). Interferon solutions in MEM-albumin (1 ml) were placed on the columns, which were eluted with MEM-albumin at a flow rate of no more than one drop per minute. Fractions of 1 ml were collected and interferon titers determined in each fraction after serial two-fold dilution. Columns onto which mouse fibroblast interferon had been loaded, were eluted with MEM-albumin first, then with 0.07 M N-acetylneuraminyl lactose at pH 2. 

One mL interferon solution (2 X 103 1U) in MEM plus 50 pg/mL bovine serum albumin was loaded onto a small column containing 0.2 mL of the Sepharose-ganglioside adduct as described in Materials and Methods. The column was first eluted with MEM-albumin alone. At arrow, elution was continued with a solution of 0.07M X-acetylneuraminyl lactose in MEM-albumin at pH 2. Antiviral activity in each fraction was determined as described in Materials and Methods. A small amount of the antiviral activity (7%) passed the column unretarded the remaining portion (89% of that applied) was eluted with fi-acetylneuraminyl lactose. 

That T-cell Interferon does not bind to gangliosides is demonstrated by its behavior on ganglioside affinity columns ... 

Under conditions where over 90% of mouse fibroblast interferon was retained (as shown in Figure k) T-cell interferon quantitatively eluted in the breakthrough of the column (Figure 8). T-cell interferon, after passage through the affinity column, was still insensitive to ganglioside inhibition, excluding the possi-... 

Figure 4. Diagrams of gangliosides of various tissues. Open column gangliosides with Fl-acetylneuraminic acid. Hatched column gangliosides with N-glycolylneu-...

Gangliosides assayed were eluted from the plate shown in Figure 3, diluted, and the amount derived from 0.1 g of brain was added to the PFC assay cultures. Values are the mean standard error of five cultures. Application of the Student t test to the standard errors for the samples gave p values less than 0.05 when compared with the Thy-l-active fraction. Positive control for the anti-Thy-1.2 PFC assay was a column GM1 fraction containing Thy-l glycolipid (15). 

Further characterization of Thy-1 active glycolipids was accomplished by fractionating brain gangliosides into mono-, di- and trisialo-gangliosides by DEAE column chromatography according to the procedure described by Nagai (14). These fractions eluted from the column were tested for Thy-1 activity (Table III). 

Table III FRACTIONATION OF BRAIN GANGLIOSIDES BY DEAE COLUMN CHROMATOGRAHPYa...

The substrate, fucosyl-GMj, was separated from its product, GMj, by chromatography on a LiChrosorb-NH2 column (4 mm X 250 mm, 5 /urn). The mobile phase was a 25 75 mixture of 5 mM potassium phosphate buffer (pH 5.5) and acetonitrile. Similarly, fucosyl-GDib was separated from GDib. except the concentration of phosphate buffer was 20 mM Gangliosides were detected by their absorbance at 195 nm. 

Figure 9.148 HPLC profile of gangliosides (A) GMt, (B) fucosyl-GMi. (C) GDib, and ( >) fucosyl-GDbi. Approximately 2 fig of each ganglioside was eluted with the buffer described in the text from the LiChrosorb-NHz column. (From Johnson, et al., 1990.)...

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