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PLANT PHYSIOLOGY , Vol 112, Issue 3 1141-1149, Copyright © 1996 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Evidence That Barley 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Kinase Is a Member of the Sucrose Nonfermenting-1-Related Protein Kinase Family
JHA. Barker, S. P. Slocombe, K. L. Ball, D. G. Hardie, P. R. Shewry and N. G. Halford
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS18 9AF, United Kingdom (J.H.A.B., S.P.S., P.R.S., N.G.H.)
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 (Mr) 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 Mr 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 Mr 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.
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