Polysialic acid is an important virulence factor for most of the capsular types of E. coli and Neisseria meningitidis causing meningitis in infants and children. This poorly immunogenic polysaccharide plays a role in neural tissue development. CMP-N-acetylneuraminic acid synthetase, a key enzyme in the formation of polysialic acid and sialylated glycoconjugates, has been purified previously in this laboratory from polysialic acid producing bacteria and the gene sequenced. Polysialylated neural adhesion molecule (N-CAM) has been detected in pituitary glands. To determine common structural features of CMP-neuAc synthetase this enzyme was purified from bovine pituitary glands. Bovine pituitary gland homogenates had a specific activity of 0.02-0.1 micro moles/min/mg protein which is equal to or greater than that detected in bovine brain homogenates, a common source for mammalian CMP-neuAc synthetase. The enzyme was partially purified to a specific activity of 20-100 micro mole/min/mg protein from soluble fractions of homogenates by ammonium sulfate fractionation, ion-exchange chromatography, dye-binding and affinity chromatography on CDP-agarose. The enzyme activity elutes on FPLC gel filtration columns with a molecular weight of 150,000, which is larger than that observed for bacterial and rat liver enzymes. The isoelectric point of partially purified enzyme based on activity was determined to be 6.5. The enzyme is sensitive to the thiol reactive reagent N-ethylmaleimide. Substrate analogs have proven to be useful tools in the study of enzyme function and the purification. Synthetic schemes for 2 types of sialic analogs have begun (a) Solid phase beta-neuAc ketosides to aid in the purification of sialic acid utilizing enzymes and (b) 9-substituted sialic acid as potential substrates for sialyl transferases. The required 6-substituted precursor derivatives of N-acetylmannosamine (6-azido and 6-iodo) to prepare the corresponding 9- substituted have been prepared. The beta-allyl ketoside of sialic acid has been prepared a precursor for coupling to a solid phase.
