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 b y phosphorylation on serine-543, followed by binding of the phosphoryl ated enzyme to 14-3-3 proteins. We purified one of several chromatogra phically 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 pe ptide sequences very similar to Arabidopsis CDPK6 (accession no. U2062 3; 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 phosp horylated NR was inhibited by 14-3-3 proteins. However, the bacteriall y expressed CDPKG had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR b y CDPK is discussed, and the regulatory properties of the purified CDP K are considered with reference to current models for reversible intra molecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs.