Adenosine Monophosphate-Activated Protein Kinase

Hydroxymethylglutaryl-CoA Reductase Kinase

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)362-371
Number of pages10
JournalMethods in Enzymology
Volume200
Issue numberC
DOIs
Publication statusPublished - 1 Jan 1991

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Hydroxymethylglutaryl CoA Reductases
Adenosine Monophosphate
Protein Kinases
Phosphotransferases
Acetyl-CoA Carboxylase
Peptides
Assays
mevalonate kinase
Rats
Phosphorylation
Liver Microsomes
Cyclic AMP-Dependent Protein Kinases
Radioisotopes
Alanine
Liver
Serine
Arginine

Cite this

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title = "Adenosine Monophosphate-Activated Protein Kinase: Hydroxymethylglutaryl-CoA Reductase Kinase",
abstract = "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.",
author = "David Carling and Clarke, {Paul R.} and Hardie, {D. Grahame}",
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Adenosine Monophosphate-Activated Protein Kinase : Hydroxymethylglutaryl-CoA Reductase Kinase. / Carling, David; Clarke, Paul R.; Hardie, D. Grahame.

In: Methods in Enzymology, Vol. 200, No. C, 01.01.1991, p. 362-371.

Research output: Contribution to journalArticle

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