Regulation of a NAD+ kinase activity isolated from asynchronous cultures of the achlorophyllous ZC mutant of Euglena gracilis.
NAD+ kinase was isolated by chromatography steps from asynchronous cultures of the achlorophyllous ZC mutant of Euglena gracilis. A non Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.
Pou de Crescenzo
M A
MA
Groupe de Biochimie et de Biologie Moléculaire Végétales, UFR Sciences, Université d'Angers, France.
Goto
K
K
Carré
I A
IA
Laval-Martin
D L
DL
D000818:Animals; D002734:Chlorophyll / Q000235:genetics; D002850:Chromatography, Gel; D002940:Circadian Rhythm; D005056:Euglena gracilis / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism*
D009243:NAD; D002734:Chlorophyll; D009249:NADP; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase