Characterization and possible redox regulation of the purified calmodulin-dependent NAD+ kinase from Lycopersicon pimpinellifolium.

ABSTRACT The soluble and calmodulin (CaM)-dependent NAD+ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short-term NaCl stress (Delumeau, Morère-Le Paven, Montrichard, Laval-Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol-modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD+ kinase. A three-step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD+ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL-1 bovine CaM. The activity of NAD+ kinase was strongly inhibited by thiol-modifying reagents and oxidized glutathione. NAD+ kinase was also found to be air-inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five-fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD+ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP-thioredoxin reductase system. [ABSTRACT FROM AUTHOR]