γ‐Phosphate‐linked ATP‐Sepharose for the affinity purification of protein kinases: Rapid purification to homogeneity of skeletal muscle mitogen‐activated protein kinase kinase

Published

Journal Article

Recently, Sowadski and colleagues [Knighton, D. R., Zheng, J., Eyck, L. F. T., Ashford, V. A., Xuong, N., Taylor, S. S. & Sowadski, J. M. (1991) Science 407, 407–420] reported the structure of a ternary complex of the catalytic subunit of cAMP‐dependent protein kinase (cyclic A kinase), 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 numerous conserved residues at the active site defined [Zheng, J., Knighton, D. R., Eyck, L. F. T., Karlsson, R., Xuong, N., Taylor, S. S. & Sowadski, J. M. (1993) Biochemistry, in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the α, β and γ phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [γ‐32P]adenosine‐5′‐(γ‐4‐aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its γ phosphate. In the presence of 20 μM ATP, adenosine‐5′‐(γ‐4‐aminophenyl)triphosphate exhibited apparent Ki values of 103.6, 75.18, 176.28 and 120.00 μM against cyclic A kinase, mitogen‐activated protein kinase (p42mapk), mitogen‐activated protein kinase kinase and p60c‐src, respectively. To illustrate the effectiveness of adenosine‐5′‐(γ‐4‐aminophenyl)triphosphate–Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen‐activated protein kinase kinase over 19000‐fold to homogeneity. Copyright © 1993, Wiley Blackwell. All rights reserved

Full Text

Duke Authors

Cited Authors

  • HAYSTEAD, CMM; GREGORY, P; STURGILL, TW; HAYSTEAD, TAJ

Published Date

  • January 1, 1993

Published In

Volume / Issue

  • 214 / 2

Start / End Page

  • 459 - 467

Electronic International Standard Serial Number (EISSN)

  • 1432-1033

International Standard Serial Number (ISSN)

  • 0014-2956

Digital Object Identifier (DOI)

  • 10.1111/j.1432-1033.1993.tb17942.x

Citation Source

  • Scopus