Tween-20 activates and solubilizes the mitochondrial membrane-bound, calmodulin dependent NAD+ finase of Avena sativa L.

Among different treatments assayed, a mix of a nonionic detergent (5% Tween-20) with 0.5 m NaCl was found to solubilize a large part of the calmodulin-dependent NAD+ kinase bound to the inner mitochondrial membrane. It also stimulated its activity by increasing 7 times the maximal velocity. Activity stimulation was also observed with phosphatidylcholine, phosphatidylethanolamine and with reductants (HSO3 and DTT). This solubilized NAD+ kinase and the calmodulin-dependent cytosoluble isoform displayed distinct molecular masses, as well as different kinetic parameters. We propose that solubilization of membrane-bound NAD+ kinase could occur in vivo in Avena sativa and could generate a soluble isoform.

Evidence of active NADP(+) phosphatase in dormant seeds of Avena sativa L.

Freshly-harvested seeds of Avena sativa L. do not germinate when imbibed at temperatures higher than 25 degrees C. This high temperature dormancy is due to the seed coats, and to the low activities of glycolysis and the oxidative pentose phosphate pathway (OPP) in the embryo. The analysis by exclusion chromatography of soluble NADP(+) phosphatase activities of embryos revealed two isoforms: a 37 kDa isoform present in both dormant and after-ripened caryopses, and a second isoform, with an apparent molecular weight of 160 kDa, five times more active in embryos of dormant seeds than in the after-ripened ones, after 6 h of imbibition at 30 degrees C. Moreover, the activity of this 160 kDa isoform was three times less in embryos from dormant caryopses when they were grown at 10 degrees C, a permissive temperature for radicle protrusion. These results suggest a correlation between the activity of the 160 kDa NADP(+) phosphatase and the dormancy state of the caryopsis. The two isoforms differed in the pH required for optimal activity: pH 5.7 and 6.5 for the 37 kDa and the 160 kDa phosphatases, respectively. Furthermore, the 160 kDa NADP(+) phosphatase displayed a strong specificity for NADP(+), whereas the 37 kDa isoform was able to hydrolyse numerous other phosphorylated compounds.

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.