Purification and characterization of the AMP‐activated protein kinase

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Abstract
1 We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2 The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonyl-benzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [γ32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3 In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 μM and 1.9 μM respectively. AMP increases the Vmax 3–5-fold without a significant change in the Km for either protein or ATP substrates. 4 The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 μM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5 These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.

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