Kinase 347
3.6. Activators
The activity of polynucleotide kinase is stimulated by sodium chlo-
ride, potassium chloride, and polyamines (22). Spermine promotes
tetramer formation (14), and potassium chloride maintains the enzyme in
this oligomeric form. A total of 1.7 mM of spermine can increase
reaction rate by 30-fold (26). Spermidine, in addition to enhancing the
reaction rate, also has the ability to inhibit nucleases present in some
kinase preparations (27).
Polyethylene glycol (PEG 8000) also improves the efficiency of the
reaction (26). Stimulation depends on the PEG concentration, which
should ideally be titrated within the range 4-10%. The stimulating
action of PEG is owing to it causing the DNA to undergo a "psi"
transition and collapse into a highly condensed state (28). This state is
only achievable for DNA molecules >300 bp in length, so PEG has
little effect on shorter molecules.
- Inhibitors
T4 polynucleotide kinase is inhibited by inorganic phosphate and
pyrophosphate (2,3,26). If 70 mM sodium or potassium phosphate
buffers (pH 7.6) are employed, enzyme activity is reduced to 5% of
that observed in Tris buffer. The reaction rate in Tris buffer can be
halved by the addition of phosphate to 7-20 mM or pyrophosphate to
5 mM (21,29). In addition, 50 mM of potassium phosphate will cause
60% inhibition of the exchange reaction (18).
Inorganic phosphate is, however, relatively more inhibitory to E.
coli alkaline phosphatase than to T4 polynucleotide kinase. It can there-
fore be used to enhance phosphorylation when phosphatase is present
(30), e.g., when end-labeling molecules after a prior phosphatasing step.
Another strong inhibitor of polynucleotide kinase is the ammonium
ion. For example, 75% inhibition can be caused by the presence of 7
mM ammonium sulfate (29,31).
Potassium chloride has an activating effect (as described in Section
3.6.), but high concentrations can be inhibitory (19). This inhibition is
not observed in substrates with protruding 5'-OH termini. Sodium
chloride, although stimulating kinase activity toward single-stranded
substrates (26), can also be inhibitory with some duplexes (21).