Mannans acetolysis

Antibodies can be used for detection of carbohydrate sequences in biological materials. For example, a mannotetraose (Manal-3Manal-2Manal-2Man) obtained by selective acetolysis of yeast mannan was derivatized with phenethylamine and diazotized to edestin (3). Specificity of the anti-mannotetraose antibodies obtained was studied by the radioimmunoassay methods just described. Results of these studies are summarized in Table II. 

Oligosaccharide Fragments Obtained Following Partial Acetolysis of Mannans Having (1— 6)-Linked a-D-Mannopyranosyl Main-Chains... 

A branched mannan extracted from the cells of the yeast Tricho-sporon aculeatum also contains a-D-(l- 2)- and a-D-(l— 6)-link-ages (see Table III). Partial acetolysis gives rise to a (I— 2)-linked mannohexaose this represents a maximum of five consecutive a-D-(I- 2)-linkages in the mannan. 

A more subtle specificity of acetolysis was suggested by Smith and Srivastava. From the results of work on the glucomannan of lies mannan, it was suggested that the linkage between two n-mannose residues is split more readily than the linkage between D-glucose and n-mannose residues. No work has since been done on simple systems to test this hypothesis. Experiments on a series of disaccharides could usefully extend our knowledge of acetolysis. 

Ballou and coworkers - studied in greater detail the structure of yeast mannan produced by S. cerevisiae grown in a culture medium containing D-glucose. They showed that controlled acetolysis by the method of Gorin and Berlin selectively cleaves (1 — 6)-linkages of this polysaccharide, and mainly produces (in excellent yield) di-, tri-, and tetra-saccharides having relatively stable (1 2)- and (1 - 3)-... 

The polysaccharide product has been respectively studied by acetolysis, exhaustive methylation, and treatment with a-D-man-nosidase. Like yeast mannan, it contains D-mannose residues linked through (1 - 2)-, (1 3)-, and (1 - 6)-linkages, but the degree... 

The second important step in elucidation of mannoprotein structure was the discovery of an enzyme with the ability to remove the mannan sidechains selectively so that the backbone structure co ild be studied (12). As a consequence, several yeast mannans, although not all, have been shown to have a linear al -linked backbone (13> 1, 1 ). As a corollary, it follows that the acetolysis oligosaccharides represent sidechain fragments of the mannan, and by their characterization we were able to arrive at schematic structures for the mannan chains (Figure 2). 

Rabbit antisera raised against intravenously injected whole yeast cells give strong precipitin reactions with isolated mannopro-teins (l6) and the homologous reactions can usually be inhibited by acetolysis fragments from the mannan (1T> l8, 195 20). Such studies revealed that the immunodominant structures of the yeast cell surface are the mannoprotein sidechains, and that some side-chains are more immunogenic than others (Figure 3). 

Controlled acetolysis which cleaves the (l->6) linkages preferentially, gives finger prints for the yeast mannan, the structure of which consists of a (l->6) linked backbone with (l->2) or (l->3) linked short side chains (] ). We have tried to use this method for the Pyricularia oryzae heteroglycan. The PHG and the Arthrobacter exo-a-D-mannanase resistant PHG core were acetolyzed for 12 h at 0°. The deacetylated acetolysis products were separated on a column of Bio-Gel P-2 (Fig. ). Typical acetolysis patterns were obtained from the original PHG. It could be seen from the elution profile that the original PHG has either a (l->6) linked backbone with (l->2) or (1- 3) linked side chains, or (l->2) or (l- 3) linked oligosaccharide blocks interspersed in a molecule and these were link-... 

Figure 8. Location of C-mannosyl residues derived from C-phosphogalacto-mannan following acetolysis (13). A reaction mixture containing 30 mg phospho-galactomannan, 125 fig of crude mannosyl-transferase, and other components of the mannosyl-transferase system (13) were incubated for 2 hr and the reaction stopped and phosphogalactomannan reisolated. Twenty mg of phosphogalactomannan was obtained. The polymer was subjected to acetolysis and the products were deacetyl-ated and fractionated on Bio-Gel P-2 as...

In yeasts particularly, chemotaxonomy by means of application of proton magnetic resonance spectroscopy was described by Gofin and Spencer(2), and the acetolysis fingerprinting technique by Kocourek and Ballou(3) can be also used to determine the taxonomy of yeasts. These methods are based on the differences in the chemical structure of their mannan components. Similarly, serological clasification of yeasts by the use of slide agglutinin test was reported by Tsuchiya, Fukazawa and Kawakita(4). 

Figure 7. Acetolysis fingerprints of Subfractions A (A), B (B), C (C), D (D), and E (E) (5), Each acetolysate, obtained from 100 mg of the mannan subfractionsy was applied onto a column (2 X 100 cm) of Bio-Gel P-2 and eluted with water (7 mL/hr). Samples (20 ixL) of eluates corresponding to Fractions 7, 11 y Illy and IV were assayed for the amounts of carbohydratey and 50 /xL of Fraction Vo was used for the determination of carbohydrate and phosphorus in the void volume regions (%) carbohydrate at 490 nm (X) phosphorus at 820 nm O.D., optical density.

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