79] Formation of type 3 pneumococcal capsular polysaccharide

Abstract

Publisher Summary This chapter discusses the formation of type 3 pneumoeoccal capsular polysaccharide. The type-specific capsular polysaccharide of Diplococcus pneumonia type 3(S3) has assigned the structure and physical measurements indicate that it is a linear polymer with a molecular weight of 171,800. Decapsulated, resting, type 3 cells will synthesize capsular polysaccharide from glucose or from certain other monosaccharides in a medium containing magnesium, potassium, and phosphate ions. It is clearly demonstrated, that a particulate fraction sedimented at 100,000 g from disintegrated type 3 cells will form type 3 polysaccharide from uridine diphosphate (UDP)-glucose and UDP-glucuronic acid. The particulate enzyme fraction is incubated in a Tris- Mg ++ buffer at pH 8.35 in the presence of UDP-glucose and UDP-glucuronic acid, the reaction is stopped by heat, and the polysaccharide synthesized is measured by the amount of antigen-antibody precipitate formed with a specific type 3 pneumococcal antiserum. The identity of the synthesized material with native S3 can be demonstrated by the 2-dimensional immune diffusion technique. All manipulations subsequent to the growth of the cells are carried out at 3°. The reagents used, procedure followed, and the steps involved in the purification are also described in the chapter.