Apparent K m, Calcium, Calcium-Calmodulin Complex, Circadian Regulation, Enzyme Regulation N AD+ kinase was isolated by chromatography steps from asynchronous cultures of the achlorophyllous ZC m utant 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 param eters revealed two kinds of N A D + binding site, depend ing on N A D + concentrations, and changes induced by EG TA , Ca2+ and Ca2+-calmodulin. The search for effectors, soluble (S) and mem brane-bound (P), in Euglena gracilis synchro nously 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 N A D + kinase activity -was also undertaken by testing the modulations of the kinetic param eters of the prepared N A D + kinase. The results suggest: (i) structural changes of N A D + binding sites depending on N A D + concentrations; (ii) possible binding of the Mg-ATP-2 (or Ca-ATP-2) on the N A D + sites, because of their common A D P motif; and (iii) different and specific modulations of the kinetic param eters of the two types of N A D + 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 N A D +; (ii) the second one, possibly the Ca2+-calmodulin complex, which provokes a com plete reverse effect. Each of these two effectors seems to be, alternatively and rhythmically (eight circadian hours apart), partially released from the membranes.
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