| Abstract: | Anionic groups were demonstrated in the mucus of Euglena gracilis Klebs var. bacillaris Cori by histochemical staining with alcian blue or diaminobenzidine tetrahydrochloride and were quantified during the growth cycle with an alcian blue dye-binding assay. Mucus in the culture increased during growth and became high when the culture entered the stationary phase. Cultures were grown under conditions which uniformly labeled all sulfur containing compounds with 35S. A purification scheme was devised using 0.15 M NaCl and 0.10 M EDTA at pH 8 (Marmur's solution) to separate the mucus from the cells without cell breakage. The isolated purified mucus was fractionated with sodium dodecyl sulfate (SDS) at 100° C into soluble and insoluble components. The soluble fraction was separated by SDS polyacrylamide gel electrophoresis into 18 polypeptide bands ranging from 22 to 320 kdaltons that stained with Coomassie blue; 16 of these bands also stained for carbohydrates using periodic acid-Schiffs reagent, indicating their glycoprotein nature. On hydrolysis, the SDS soluble fraction yielded xylose, fucose, rhamnose, and hexose. The SDS insoluble fraction contained no 35S label, and, therefore, presumably no protein or bound sulfate; this gelatinous material does not contain the same sugar residues as the glycoproteins in the SDS soluble fraction. Its staining properties with alcian blue and its resistance to hydrolysis suggested the presence of uronic acids. Comparison with other Euglena fractions showed that bands comigrating with the mucus glycoproteins were not detectable in the fractions containing the whole cells or the culture medium. Although the mucus of Euglena yielded appreciable sulfate during mild acid treatment, most if not all of this sulfate appears to have come from the oxidation of reduced sulfur rather than from the hydrolysis of covalently bound sulfate. An infrared spectrum of the mucus showed only minor peaks in the correct regions for the S-O linkage. Thus, the mucus of Euglena is composed of glycoproteins and polysaccharides which contain little or no ester sulfate. |
