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Purification and characterization of polyphosphate kinase from Neisseria meningitidis
C R Tinsley1, B N Manjula, E C Gotschlich
1Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University, New York, New York 10021-6399.
Abstract:
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 [gamma-32P]ATP 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.
Insights
Researchers purified polyphosphate kinase (PPK) from Neisseria meningitidis to study polyphosphate
Area of Science:
- Microbiology
- Biochemistry
Background:
- Neisseria meningitidis and Neisseria gonorrhoeae are human pathogens that store phosphate as polyphosphate.
- Extracellular polyphosphate may function as a protective capsule in Neisseria species.
Purpose of the Study:
- To enable genetic investigation into the role of polyphosphate in Neisseria species.
- To purify and characterize the enzyme polyphosphate kinase (PPK) from Neisseria meningitidis.
Main Methods:
- Purification of polyphosphate kinase (PPK) from Neisseria meningitidis BNCV.
- Enzyme activity assays dependent on Mg2+, phosphate/polyphosphate, and ATP.
- Characterization of kinetic parameters (Km, turnover number) and enzyme phosphorylation using [gamma-32P]ATP.
- Amino acid sequencing of the purified PPK protein.
Main Results:
- Polyphosphate kinase (PPK) was successfully purified from Neisseria meningitidis.
- PPK activity requires Mg2+ and phosphate/polyphosphate and is inhibited by ADP.
- Kinetic analysis revealed a Km for ATP of 1.5 mM and a turnover number of 47 phosphate residues/polypeptide/s.
- The enzyme is phosphorylated during the reaction, likely at an arginine residue.
- N-terminal and internal amino acid sequences were obtained for PPK.
Conclusions:
- The characterization of Neisseria meningitidis PPK provides a foundation for its genetic manipulation.
- Understanding PPK's role is crucial for investigating polyphosphate's function in Neisseria pathogenesis.
- The derived amino acid sequences will facilitate cloning and genetic studies of the ppk gene.