Precursor-Product Relationships during Sulfate Incorporation into Porphyridium Capsular Polysaccharide

This study describes the kinetics of (35)S-incorporation during in vivo sulfate esterification of Porphyridium aerugineum capsular polysaccharide. Techniques were developed to isolate the precursor pool (free sulfate), cell-associated product, and extracellular product. Specific radioactivities of these three fractions were monitored during pulse-chase sequences. Label rapidly appeared in the pool during the pulse, then declined asymptotically during the chase as equilibrium was approached. Efflux of small quantities of isotope from the cell during chase periods was not the result of backleakage, but the result of washing untransported isotope from the free-space. During the pulse, intracellular product was labeled at 25% of the rate at which the pool was labeled. Fully 50% of the label which left the pool was incorporated into the polysaccharide as ester sulfate, indicating that polysaccharide esterification is a major metabolic pathway for sulfate. The specific radioactivity of the extracellular product increased slowly throughout pulse and chase periods.Porphyridium was shown to be highly dependent on exogenous supplies of sulfate, the cells lysing when denied adequate quantities of this nutrient. The free sulfate pool size was measured as 33 x 10(6) sulfate ions per cell in log phase. 3'-Phosphoadenosine-5'-phosphosulfate was tentatively identified in the water-soluble cell extract and is thought to be the "activitated" donor for sulfate transfer reactions.