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

Open Access
Publisher Website
Google Scholar
Abstract
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 [γ‐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.