Identification of an endogenous inhibitor of AMP-activated protein kinase.

AMP-activated protein kinase (AMP-PK) is a multi substrate kinase with an important role in the regulation of mammalian lipid metabolism. The enzymes that regulate fatty acid and cholestrol biosynthesis are acetyl-CoA carboxylase and 3hydroxymethylglutaryl-CoA reductase, respectively. Hormonal regulation of each is achieved through their phosphorylation and inactivation by AMP-PK. Like many other serine kinases, AMP-PK is allostaically activated by a small molecular weight ligand (in this case pM 5’ AMP) and is itself phosphorylated and activated by a distinct kinase kinase [ I , 21. In addition to these regulatory mechanisms, many protein kinases have specific endogenous protein or peptide inhibitors that are pseudosubstrates (ie. contain the protein kinase consensus recognition sequence but lack the target senne). For example, Walsh inhibitor is a heat-stable, 12KDa endogenous protein that is a pseudosubstrate for cyclic AMPdependent protein kinase [3]. Similar but less well characterised protein and peptide inhibitors exist for other protein kinases such as protein lrinase C [4,5]. The dilution of crude tissue extracts for the assay of AMP-PK activity often leads anomalously to increased activity which suggests that an inhibitor may have been diluted away [6, MRM unpublished]. This study has identified an inhibitor of AMP-PK in rat liver and skeletal muscle. We have parhally purified these inhibitory activities using a 2040% ammonium sulphate precipitation and application of the resuspended protein pellet to a DE52 anion exchange column on FPUJ followed by elution with 35OmM NaCI. The achvity of the inhibitor was assessed by its ability to inhibit the incorporation of 32P from [y-32P]-ATP into the specific peptide substrate HMRSAMSLHLVKRR (based on the phosphorylation site on acetylCoA cahxylase) by purified rat liver AMP-PK. A full description of the AMP-PK assay is given in 171. Some p r o v e s of the inhibitory activities of the post-DE52 fractions from both liver and skeletal muscle have been investigated. Both inhibited AMP-PK in a concentration-dependent manner. Full inhibition was not time-dependent but observed within the 5 minutes assay time for AMP-PK activity. Typical levels of inhibition seen were 90% from liver, and 2040% from muscle preparations. However, both preparations are considerably impure (confirmed by SDS-PAGE) so that one could not compare the specific activities of muscle and liver inhibitors