Evidence that barley 3-hydroxy-3-methylglutaryl-coenzyme a reductase kinase is a member of the sucrose nonfermenting-1-related protein kinase family

J H Barker, S P Slocombe, K L Ball, D G Hardie, P R Shewry, N G Halford

Research output: Contribution to journalArticlepeer-review

Abstract

A protein kinase was partially purified from barley (Hordeum vulgare L. cv Sundance) endosperm by ammonium sulfate fractionation, followed by ion-exchange, Reactive Blue, Mono-Q, and phosphocellulose chromatography. It was shown to phosphorylate Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and a synthetic peptide that was shown previously to act as a substrate for HMG-CoA reductase kinase purified from cauliflower, confirming it to be barley HMG-CoA reductase kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation showed the presence of a polypeptide with an approximate relative molecular weight (M(r)) of 60,000, which is the size predicted for the barley sucrose nonfermenting-1 (SNF1)-related protein kinases BKIN2 and BKIN12. Antisera were raised to a rye (Secale cereale L.) SNF1-related protein kinase (RKIN1) expressed in Escherichia coli as a fusion with maltose-binding protein and to a synthetic peptide with a sequence that is conserved in, and specific to, plant members of the SNF1-related protein kinase family. The maltose-binding protein-RKIN1 fusion protein antiserum recognized a doublet of polypeptides with an approximate M(r), of 60,000 in crude endosperm extracts and a single polypeptide in root extracts, which co-migrated with the smaller polypeptide in the endosperm doublet. Both antisera recognized a polypeptide with an approximate M(r) of 60,000 in the partially purified protein kinase preparation, suggesting strongly that barley HMG-CoA reductase kinase is a member of the SNF1-related protein kinase family.
Original languageEnglish
Pages (from-to)1141-9
Number of pages9
JournalPlant physiology
Volume112
Issue number3
Publication statusPublished - Nov 1996

Keywords

  • AMP-Activated Protein Kinases
  • Amino Acid Sequence
  • Animals
  • Brassica
  • Cloning, Molecular
  • Drosophila
  • Escherichia coli
  • Hordeum
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Multienzyme Complexes
  • Plant Proteins
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • Recombinant Proteins
  • Secale cereale
  • Sequence Homology, Amino Acid

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