Search

NAD+ kinase was isolated by chromatography steps from asynchronous cultures of the achlorophyllous ZC mutant of Euglena gracilis. A non Ca2+-calmodulin dependent form, whose activity was stimulated by EGTA, was selected for its large quantity and high specific activity. Studies of the kinetic parameters revealed two kinds of NAD+ binding site, depending on NAD+ concentrations, and changes induced by EGTA, Ca2+ and Ca2+-calmodulin. The search for effectors, soluble (S) and membrane-bound (P), in Euglena gracilis synchronously grown (in a light-dark regime of 12h:12h), and collected at circadian times (CT) - corresponding to the maximum, CT 17, and to the trough, CT 09, of the circadian rhythm of NAD+ kinase activity - was also undertaken by testing the modulations of the kinetic parameters of the prepared NAD+ kinase. The results suggest: (i) structural changes of NAD+ binding sites depending on NAD+ concentrations; (ii) possible binding of the Mg-ATP-2 (or Ca-ATP-2) on the NAD+ sites, because of their common ADP motif; and (iii) different and specific modulations of the kinetic parameters of the two types of NAD+ binding site by the Ca2+-calmodulin complex. In addition, the results indicate, in pelletable fractions isolated at CT 09 and CT 17, the presence of two kinds of effector: (i) the first one, possibly Ca2+, which increases the Umax’s while decreasing the binding of NAD+; (ii) the second one, possibly the Ca2+-calmodulin complex, which provokes a complete reverse effect. Each of these two effectors seems to be, alternatively and rhythmically (eight circadian hours apart), partially released from the membranes