PURIFICATION OF THE AMP-ACTIVATED PROTEIN-KINASE ON ATP-GAMMA-SEPHAROSE AND ANALYSIS OF ITS SUBUNIT STRUCTURE

The AMP-activated protein kinase has been purified by affinity chromat ography on ATP-gamma-Sepharose. A proportion of the activity can be el uted using AMP, while the remainder is eluted using ATP. The-AMP eluat e contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was p reviously identified as the AMP-binding catalytic subunit [Carling, D. , Clarke, P. R., Zammit, V. A. and Hardie, D. G. (1989) Eur J. Biochem . 186, 129-136]. All three polypeptides exactly comigrate both on nati ve gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4-5.8 nm) by gel filtration, and sedimentation coefficient (7.9-8.4 S) by glycer ol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 +/- 10 k Da. Thus the native enzyme appears to be a heterotrimer with a p63/p38 /p35 (1:1:1) structure. Despite the fact that the ATP eluate has a hig her specific activity than the AMP eluate (3.5 +/- 0.2 vs 2.3 +/- 0.2 mu mol min(-1) mg(-1)), it appears to be less pure, containing p63, p3 8 and p35 plus other polypeptides. Experiments examining the effects o f protein phosphatase-2A and kinase kinase, and analysis by Western bl otting with anti-p63 antibody, suggests that the AMP eluate is entirel y in the low-activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high-activity phosphorylated form. As we ll as establishing the subunit structure of the AMP-activated protein kinase, these results suggest that the kinase can bind to ATP-gamma-Se pharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases th e affinity for the latter site.