A pull-down assay for 5' AMP-activated protein kinase activity using the GST-fused protein.
An assay using a specific peptide (SAMS peptide) as a substrate is widely used for determination of AMP-activated protein kinases (AMPK) activity. However, it is not an efficient assay for crude AMPK preparations. In this study, we modified the assay by using the SAMS peptide fused to glutathione-S-transferase (GST-SAMS) instead of the SAMS peptide on its own. Radioactivity incorporated into GST-SAMS can be recovered easily by precipitation with glutathione-agarose. The kinetic parameters of partially purified AMPK for the GST-SAMS were as follows. The Vmax was 0.26 +/- 0.012 nmol/min/mg of total proteins and Km for GST-SAMS was 110 +/- 12 microM. The parameters for ATP were 0.40 +/- 0.016 nmol/min/mg of total proteins (Vmax) and 202 +/- 21 microM (Km). The activity of AMPK in this system was stimulated about threefold by the AMPK activators, AMP or 5-amino-4-imidazolecarboxamide ribotide (ZMP), and inhibited by the AMPK inhibitors, adenine 9-beta-D-arabinofuranoside (ara-A) and iodotubercidin. These values correlate well with those for the SAMS peptide reported previously. Thus, we successfully established a convenient and rapid method to measure AMPK applicable, even for crude enzyme preparations.
Kishimoto
Atsuhiro
A
Cancer Physiology Project, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan.
Ogura
Tsutomu
T
Esumi
Hiroyasu
H
D055372:AMP-Activated Protein Kinases; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D013379:Substrate Specificity; D014372:Tubercidin / Q000031:analogs & derivatives / Q000494:pharmacology; D014740:Vidarabine / Q000494:pharmacology
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; D014740:Vidarabine; D014372:Tubercidin