Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase |
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Authors: | Pauline Douglas Greg Moorhead Yan Hong Nick Morrice Carol MacKintosh |
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Institution: | (1) Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 5EH, UK, GB;(2) Institute of Molecular Agrobiology, 59A The Fleming, 1 Science Park Drive, Singapore 118240, Republic of Singapore, SG |
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Abstract: | Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme
to 14-3-3 proteins. We purified one of several chromatographically distinct NRserine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent
(calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated
NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower
than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties
of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like
domain to the autoinhibitory junction of CDPKs.
Received: 12 February 1998 / Accepted: 28 May 1998 |
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Keywords: | Key words: Calmodulin-domain protein kinase Nitrate reductase Phosphorylation 14-3-3 proteins Spinacea (nitrate reductase) |
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