Characterisation of 5'-AMP-activated protein kinase in human liver using specific peptide substrates and the effects of 5'-AMP analogues on enzyme activity.
A specific peptide (SAMS peptide) phosphorylation assay has previously been used to measure and subsequently purify rat liver 5'-AMP-activated protein kinase (AMPK). In this report, we show that this peptide and a peptide based on the sequence surrounding the site phosphorylated on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase by AMPK (HMG peptide) can be used to measure human liver AMPK. Our data demonstrate that both human and rat AMPKs have a higher affinity for the HMG peptide compared to the SAMS peptide. We have used these peptide phosphorylation assays to identify novel activators of AMPK.
Sullivan
J E
JE
Cardiovascular and Metabolism Research Department, ZENECA Pharmaceuticals, Alderley Park, Cheshire, U.K.
Carey
F
F
Carling
D
D
Beri
R K
RK
D055372:AMP-Activated Protein Kinases; D000227:Adenine Nucleotides / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D013329:Structure-Activity Relationship
D000227:Adenine Nucleotides; D009097:Multienzyme Complexes; D010455:Peptides; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases