experience this assay is preferred for enzyme kinetic experiments
since it can be easily adapted for testing at a range of peptide or ATP
concentrations [9, 10, 13]. Once the phosphorylation reaction is
completed, it is quenched with phosphoric acid at desired time
points. Under these conditions, the kinase is inactivated, the
SAMS peptide becomes protonated allowing for retention on the
phosphocellulose membrane via a charge interaction, and the^33 P-
ATP is removed by filtration. The method below describes a dis-
continuous assay format where a reaction progress curve is gener-
ated by measuring the amount of phosphorylated SAMS peptide
product produced over time. The linear range of each time course
can be used to determine the rate at each reaction condition. The
rates determined at different substrate concentrations can then be
plotted as a function of (S) to elucidate the Km and Vmax
parameters [9].1.5 Surface Plasmon
Resonance
Surface plasmon resonance (SPR) is the biophysical technique of
choice in pharmaceutical research to determine equilibrium bind-
ing affinities (KD) and binding kinetics (kon,koff) of small molecules
to protein targets [27]. In this method, protein reagents, referred
to as the ligand in SPR, are immobilized onto a sensor chip using
direct immobilization protocols such as amine coupling of surface
lysines or by an interaction of an engineered protein tag to a specific
chip surface. For example, this type of immobilization could
include coupling of a His-tagged protein onto a Ni-NTA surface
or capture of a biotin-tagged protein on a streptavidin surface, as is
described below for AMPK. In SPR, binding affinities and kinetics
are determined by injecting the small molecule, referred to as the
analyte, over the sensor surface containing immobilized protein.
The analyte is usually prepared in aqueous sample buffer. Binding
of an analyte to the immobilized protein causes a change in the
refractive index at the sensor surface, which is captured by a sensi-
tive detector.2 Materials
2.1 Recombinant
Protein Production
- Vector for expression in bacterial cells: pET14b expression
vector. - Host cells for bacterial expression:E. coliBL21-CodonPlus™
(DE3)-RIPL strain. - AMPK plasmid for expression inE. coli: Tricistronic construct
that contains open reading frames of humanα,β, andγsub-
units codon-optimized for expression inE. coliwithNcoIand
XhoIsites for ligation to pET14b vector (seeNote 2). - AMPK plasmids for expression in insect cells: Genes for human
AMPKα,β, andγsubunits custom-synthesized with codon
32 Ravi G. Kurumbail et al.