GAMMA-PHOSPHATE-LINKED ATP-SEPHAROSE FOR THE AFFINITY PURIFICATION OFPROTEIN-KINASES - RAPID PURIFICATION TO HOMOGENEITY OF SKELETAL-MUSCLE MITOGEN-ACTIVATED PROTEIN-KINASE KINASE
Cmm. Haystead et al., GAMMA-PHOSPHATE-LINKED ATP-SEPHAROSE FOR THE AFFINITY PURIFICATION OFPROTEIN-KINASES - RAPID PURIFICATION TO HOMOGENEITY OF SKELETAL-MUSCLE MITOGEN-ACTIVATED PROTEIN-KINASE KINASE, European journal of biochemistry, 214(2), 1993, pp. 459-467
Recently, Sowadski and colleagues [Knighton, D. R., Zheng, J., Eyck, L
. F. T., Ashford, V. A., Xuong, N., Taylor, S. S. & Sowadski, J. M. (1
991) Science 407, 407-420] reported the structure of a ternary complex
of the catalytic subunit of cAMP-dependent protein kinase (cyclic A k
inase), MgATP and a 20-residue inhibitor peptide, at a resolution of 0
.27 nm. This structure has since been refined to 0.2-nm resolution and
the orientation of the nucleotide and interactions of MgATP with nume
rous conserved residues at the active site defined [Zheng, J., Knighto
n, D. R., Eyck, L. F. T., Karlsson, R., Xuong, N., Taylor, S. S. & Sow
adski, J. M. (1993) Biochemistry, in the press). These studies reveale
d that the adenosine portion of ATP is buried deep within the catalyti
c cleft, with the alpha, beta and gamma phosphates protruding towards
the opening of the cleft. The unique spatial positioning of MgATP with
in the catalytic cleft of cyclic A kinase and its interactions with co
nserved amino acids found in all protein kinases, led us to reconsider
the use of ATP as an affinity ligand for the purification of these en
zymes. In this paper, we describe a straightforward method for the syn
thesis of -32]adenosine-5'-(gamma-4-aminophenyl)triphosphate for the c
ovalent linkage of ATP to Sepharose through its gamma phosphate. In th
e presence of 20 muM ATP, adenosine-5'-(gamma-4-aminophenyl)triphospha
te exhibited apparent K(i) values of 103.6, 75.18, 176.28 and 120.00 m
uM against cyclic A kinase, mitogen-activated protein kinase (p42mapk)
, mitogen-activated protein kinase kinase and p60c-src, respectively.
To illustrate the effectiveness of ine-5'-(gamma-4-aminophenyl)triphos
phate-Sepharose as an affinity column for protein kinases, we have use
d the resin to purify rabbit skeletal muscle mitogen-activated protein
kinase kinase over 19000-fold to homogeneity.