Routine protein sequence analysis below ten picomoles: one sequencing
- D. Atherton, J. Fernandez, M. DeMott, L. Andrews, S. M. Mische
- facility's approach,
The important human pathogens Neisseria meningitidis and Neisseria gonorrhoeae accumulate phosphate in the form of polyphosphate (A. Noegel and E. C. Gotschlich, J. Exp. Med. 157:2049-2060, 1983), and the localization of more than half of this long-chain polymer on the exterior of the cells suggests a function as a protective, capsule-like coating. To enable further genetic investigation of the role of polyphosphate in Neisseria spp., the enzyme polyphosphate kinase (PPK), which catalyzes the synthesis of polyphosphate from ATP, was purified from N. meningitidis BNCV. The activity is dependent on Mg2+ and phosphate or polyphosphate and is inhibited by ADP. The Km for ATP is 1.5 mM, and the turnover number is 47 phosphate residues per polypeptide per s. Analysis of PPK labelled with [y_32PJATP indicates that the enzyme is phosphorylated during the reaction, probably at an arginine residue. N-terminal and two internal amino acid sequences were derived from the purified protein and will allow the design of synthetic oligonucleotides for cloning and genetic manipulation of the ppk gene.