Adenosine Monophosphate-Activated Protein Kinase: Hydroxymethylglutaryl-CoA Reductase Kinase

TY - JOUR

T1 - Adenosine Monophosphate-Activated Protein Kinase

T2 - Hydroxymethylglutaryl-CoA Reductase Kinase

AU - Carling, David

AU - Clarke, Paul R.

AU - Hardie, D. Grahame

PY - 1991/1/1

Y1 - 1991/1/1

N2 - Hydroxymethylglutaryl-CoA (HMG-CoA) reductase kinase has been previously assayed by its ability to inactivate HMG-CoA reductase in crude rat liver microsomes, using a radioisotopic assay for HMG-CoA reductase. However, this assay is insensitive and time consuming and it uses expensive radioisotopes and is subject to interference by HMG-CoA lyase and mevalonate kinase. This chapter presents a completely specific and more convenient assay involving phosphorylation of a synthetic peptide (SAMS peptide) with the sequence HMRSAMSGLHLVKRR. This peptide is based on the sequence from His-73 to Lys-85 in rat acetyl-CoA carboxylase, except that the fifth residue is alanine rather than serine, to abolish a site (corresponding to Set-77) phosphorylated by cyclic adenosine monophosphate (AMP)-dependent protein kinase. In addition, two arginines are added at the C terminus to make the peptide bind tightly to phosphocellulose paper. The AMP-activated protein kinase phosphorylates the peptide on the seventh residue, corresponding to Set-79 on acetyl-CoA carboxylase, the site which is phosphorylated most rapidly by the kinase and which appears to regulate acetyl-CoA carboxylase activity.

AB - Hydroxymethylglutaryl-CoA (HMG-CoA) reductase kinase has been previously assayed by its ability to inactivate HMG-CoA reductase in crude rat liver microsomes, using a radioisotopic assay for HMG-CoA reductase. However, this assay is insensitive and time consuming and it uses expensive radioisotopes and is subject to interference by HMG-CoA lyase and mevalonate kinase. This chapter presents a completely specific and more convenient assay involving phosphorylation of a synthetic peptide (SAMS peptide) with the sequence HMRSAMSGLHLVKRR. This peptide is based on the sequence from His-73 to Lys-85 in rat acetyl-CoA carboxylase, except that the fifth residue is alanine rather than serine, to abolish a site (corresponding to Set-77) phosphorylated by cyclic adenosine monophosphate (AMP)-dependent protein kinase. In addition, two arginines are added at the C terminus to make the peptide bind tightly to phosphocellulose paper. The AMP-activated protein kinase phosphorylates the peptide on the seventh residue, corresponding to Set-79 on acetyl-CoA carboxylase, the site which is phosphorylated most rapidly by the kinase and which appears to regulate acetyl-CoA carboxylase activity.

UR - https://www.scopus.com/pages/publications/0026356039

U2 - 10.1016/0076-6879(91)00153-N

DO - 10.1016/0076-6879(91)00153-N

M3 - Article

C2 - 1683469

AN - SCOPUS:0026356039

SN - 0076-6879

VL - 200

SP - 362

EP - 371

JO - Methods in Enzymology

JF - Methods in Enzymology

IS - C

ER -