(ACC). This peptide was modified to omit phosphorylation by
cyclic AMP-dependent protein kinase and called the SAMS peptide
from the internal sequence of the peptide
(HMRSAMSGLHLVKRR) containing the target serine residue
phosphorylated by AMPK [3–5].
In an effort to define the recognition motif of mammalian,
higher plant, and yeast members of the SNF1 protein kinase sub-
family, the synthetic peptide AMARA (AMARAASAAALARRR)
was developed and was shown to be a better substrate for AMPK
than the SAMS peptide [6]. After this discovery, an increasing
number of research groups exchanged SAMS for AMARA peptide
in activity measurements of AMPK.
In 1996, the first antibodies against theαsubunit isoforms
were generated, and AMPK could be purified with immunoprecip-
itation further increasing the specificity of the activity assay [7]. Fur-
thermore, the activity of AMPK could now be divided intoα1 and
α2 activities showing diverse regulation in skeletal muscle [8, 9]. In
2005, our research group identified the human heterotrimeric
complex composition in the skeletal muscle [10] and, shortly
thereafter, started to measure not only theα1 andα2 activities
but also theγ3-associated activity [11].
Some considerations should be made when measuring the
kinase activity of AMPK. First of all, the assay is depending on
immunoprecipitation and the quality of this as described in
Chapter 13. This raises demands to the homogenization procedure
and the IP procedure itself. During the kinase reaction, the immu-
noprecipitated AMPK heterotrimeric complex is still bound by the
antibody and the Protein G agarose beads. The antibody has
approximately the same molecular size as the whole AMPK hetero-
trimer complex, and depending on the location of the epitope, this
could potentially inhibit (or less likely increase) the activity of
AMPK. Therefore, comparison of activities between different com-
plexes (precipitated with different antibodies) or between different
laboratories using different sources of antibodies should be done
with caution.
Secondly, the AMPK activity is measured in vitro under condi-
tions that are different from the in vivo environment inside the cell.
The nucleotide status (AMP, ADP, and ATP conc.) in the cell at the
time of harvest has tremendous effects on the activity of AMPK
both in terms of allosteric and covalent (Thr-172 phosphorylation)
activation. The kinase activity assay is run with a saturated concen-
tration of AMP (200μM), and thus the direct allosteric effect of the
intracellular AMP/ATP ratio is lost in the assay. The allosteric
activation of AMPK is fortunately relatively small compared to the
covalent (Thr-172 phosphorylation), and thus it may not make a
substantial difference that the assay is run with a saturated concen-
tration of AMP. In theory, the activity measured should therefore
be equivalent to the Thr-172 phosphorylation status of the assayed216 Jesper B. Birk and Jørgen F. P. Wojtaszewski