Biochemical characterization of two forms of 3-hydroxy-3-methylglutaryl-CoA reductase kinase from cauliflower (Brassica oleracia).
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.[1]References
- Biochemical characterization of two forms of 3-hydroxy-3-methylglutaryl-CoA reductase kinase from cauliflower (Brassica oleracia). Ball, K.L., Dale, S., Weekes, J., Hardie, D.G. Eur. J. Biochem. (1994) [Pubmed]
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