Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase.
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.
Department of Biochemistry, University of Dundee, UK. pdouglas@bad.dundee.ac.uk
P
P
Douglas
G
G
Moorhead
Y
Y
Hong
N
N
Morrice
C
C
MacKintosh
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D010766:Phosphorylation; D018724:Spinacia oleracea / Q000201:enzymology*
D009565:Nitrate Reductases; D050901:Nitrate Reductase; D017871:Calcium-Calmodulin-Dependent Protein Kinases