Biochemical characterization of two forms of 3-hydroxy-3-methylglutaryl-CoA reductase kinase from cauliflower (Brassica oleracia)

K L Ball, S Dale, J Weekes, D G Hardie

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

We recently reported the existence of a protein kinase cascade in higher plants, of which the central component is a 3-hydroxy-3-methylglutaryl(HMG-)-CoA reductase kinase functionally related to mammalian AMP-activated protein kinase [MacKintosh, R. W., Davies, S. P., Clarke P. R., Weekes, J., Gillespie, S. G., Gibb, B. J. & Hardie, D. G. (1992) Eur. J. Biochem. 209, 923-931]. We have now purified this protein kinase 9000-fold from cauliflower inflorescences. During the course of this work we noticed a second minor form (form B) which separated from the major form (A) on ion exchange and gel filtration. Both forms phosphorylate the catalytic fragment of mammalian HMG-CoA reductase. Both forms are markedly inactivated by incubation with the reactive ATP analogue p-fluorosulphonylbenzoyl adenosine (FSO2PhCOAdo), and also by mammalian protein phosphatase 2C, indicating that form B, like form A, is activated by phosphorylation. Form A has an apparent native molecular mass of 200 kDa by gel filtration and, after labelling with [14C]FSO2PhCOAdo, of 150 kDa by electrophoresis in non-denaturing gels. The catalytic subunit was identified as a polypeptide of 58 kDa after labelling with [14C]FSO2PhCOAdo. Form B has an apparent native molecular mass of 45 kDa by gel filtration, and was identified as a polypeptide of 45 kDa after labelling with [14C]FSO2PhCOAdo and [gamma-32P]ATP. Using a series of variants of the synthetic peptide substrate, the substrate specificities of the two forms are similar but not identical. Form B does not appear to be a proteolytic fragment of form A, and we therefore propose that it represents a closely related member of the same protein kinase sub-family.
Original languageEnglish
Pages (from-to)743-50
Number of pages8
JournalEuropean Journal of Biochemistry
Issue number3
Publication statusPublished - 1 Feb 1994

Keywords / Materials (for Non-textual outputs)

  • AMP-Activated Protein Kinases
  • Adenosine
  • Amino Acid Sequence
  • Animals
  • Brassica
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Hydroxymethylglutaryl CoA Reductases
  • Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent
  • Liver
  • Molecular Sequence Data
  • Molecular Weight
  • Multienzyme Complexes
  • Oligopeptides
  • Phosphoprotein Phosphatases
  • Phosphorylation
  • Protein Conformation
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • Rats
  • Substrate Specificity


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