Ref ID : 4564
Marie-Anne Pou de Crescenzo, Ken Goto, Isabelle A. Carre, and Danielle Laval-Martin; Regulation of a NAD+ Kinase Activity Isolated from Asynchronous Cultures of the Achlorophyllous ZC Mutant of Euglena gracilis. Z.Naturforsch. 52c:623-635, 1997
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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 12hr:12hr), 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 Vmax'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.