Purification and characterization of the AMP‐activated protein kinase: Copurification of acetyl‐CoA carboxylase kinase and 3‐hydroxy‐3‐methylglutaryl‐CoA reductase kinase activities

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. 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. In the absence of AMP the purified kinase has apparent K_m values for ATP and acetyl‐CoA carboxylase of 86 μM and 1.9 μM respectively. AMP increases the V_max 3–5‐fold without a significant change in the K_m for either protein or ATP substrates. 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. 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.