title: Evidence that barley 3-hydroxy-3-methylglutaryl-coenzyme a reductase kinase is a member of the sucrose nonfermenting-1-related protein kinase family.
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.
authors.affiliation: IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, United Kingdom.
authors.forename: J H
authors.initials: JH
authors.lastname: Barker
authors.forename: S P
authors.initials: SP
authors.lastname: Slocombe
authors.forename: K L
authors.initials: KL
authors.lastname: Ball
authors.forename: D G
authors.initials: DG
authors.lastname: Hardie
authors.forename: P R
authors.initials: PR
authors.lastname: Shewry
authors.forename: N G
authors.initials: NG
authors.lastname: Halford
mesh_terms: D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001937:Brassica / Q000201:enzymology; D003001:Cloning, Molecular; D004330:Drosophila; D004926:Escherichia coli; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D010940:Plant Proteins; D011494:Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012434:Secale / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
chemical_list: D009097:Multienzyme Complexes; D010940:Plant Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; C081179:BKIN12 protein, Hordeum vulgare; C103365:BKIN2 protein, Hordeum vulgare; C092997:RKIN1 protein, Secale cereale; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases