Isolation and partial characterization of apiogalacturonans from the cell wall of Lemna minor

Abstract

1. A mild, reproducible extraction procedure, using 0.5% ammonium oxalate, was developed for the isolation of polysaccharides containing d-apiose from the cell wall of Lemna minor. On a dry-weight basis the polysaccharide fractions extracted with ammonium oxalate made up 14% of the material designated cell walls and contained 20% of the d-apiose originally present in the cell walls. The cell walls, as isolated, contained 83% of the d-apiose present in L. minor. 2. After extraction with ammonium oxalate, purified polysaccharides were obtained by DEAE-Sephadex column chromatography and by fractional precipitation with sodium chloride. With these procedures the material extracted at 22°C could be separated into at least five polysaccharides. On a dry-weight basis two of these polysaccharides made up more than 50% of the material extracted at 22°C. There was a direct relationship between the d-apiose content of the polysaccharides and their solubility in sodium chloride solutions; those of highest d-apiose content were most soluble. 3. All the polysaccharides isolated appeared to be of one general type, namely galacturonans to which were attached side chains containing d-apiose. The d-apiose content of the apiogalacturonans varied from 7.9 to 38.1%. The content of esterified d-galacturonic acid residues in all apiogalacturonans was low, being in the range 1.0–3.5%. Hydrolysis of a representative apiogalacturonan with dilute acid resulted in the complete removal of the d-apiose with little or no degradation of the galacturonan portion. 4. Treatment of polysaccharide fractions with pectinase established that those of high d-apiose content and soluble in m-sodium chloride were not degraded, whereas those of low d-apiose content and insoluble in m-sodium chloride were extensively degraded. When the d-apiose was removed from a typical pectinase-resistant polysaccharide, the remainder of the polysaccharide was readily degraded by this enzyme. 5. Periodate oxidation of representative polysaccharide fractions and apiogalacturonans and determination of the formaldehyde released showed that about 50% of the d-apiose molecules were substituted at either the 3- or the 3′-position.