The hypothesis that overexpression of glutamate-cysteine ligase (GCL), which catalyzes the rate-limiting reaction in de novo glutathione biosynthesis, could extend life span was tested in the fruit fly, Drosophila melanogaster. The GAL4-UAS binary transgenic system was used to generate flies overexpressing either the catalytic (GCLc) or modulatory (GCLm) subunit of this enzyme, in a global or neuronally targeted pattern. The GCL protein content of the central nervous system was elevated dramatically in the presence of either global or neuronal drivers. GCL activity was increased in the whole body or in heads, respectively, of GCLc transgenic flies containing global or neuronal drivers. The glutathione content of fly homogenates was increased by overexpression of GCLc or GCLm, particularly in flies overexpressing either subunit globally, or in the heads of GCLc flies possessing neuronal drivers. Neuronal overexpression of GCLc in a long-lived background extended mean and maximum life spans up to 50%, without affecting the rate of oxygen consumption by the flies. In contrast, global overexpression of GCLm extended the mean life span only up to 24%. These results demonstrate that enhancement of the glutathione biosynthetic capability, particularly in neuronal tissues, can extend the life span of flies, and thus support the oxidative stress hypothesis of aging.

We report an Mg 2+ -dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.z 1998 Federation of European Biochemical Societies.

One of the major protein kinases (PK III ) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, we have developed a fluorescence-based continuous assay for measurement of PK III activity. Using the continuous assay, along with the fixed-time-point 32 P-incorporation assay, we demonstrate that PK III activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8.5 to 5.5 and by reducing the concentration of Mg 2ϩ in the assay from 10 to 2 mm. Under likely physiological conditions (pH 7.0 and 2 mm Mg 2ϩ ), 10 mm Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK III in the following order: Glc-6-P Ͼ mannose-6-P, fructose-1,6P 2 Ͼ ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK III activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.Suc-P synthase (SPS) is a highly regulated enzyme that catalyzes the penultimate reaction in Suc synthesis in plants. One of the mechanisms for the regulation of SPS activity involves reversible protein phosphorylation. SPS is known to be phosphorylated on multiple seryl residues, with three of the sites potentially of regulatory significance (Huber and Huber, 1996; Toroser and Huber, 1997). The first site to be identified was Ser-158 of spinach SPS, which is the major site responsible for light/dark modulation of SPS activity (McMichael et al., 1993; Toroser et al., 1999). Ser-158 is potentially phosphorylated by several leaf protein kinases, one of which (the PK III enzyme) (McMichael et al., 1995; Toroser and Huber, 1997) is now known to be a SNF1-related protein kinase (SnRK1) (Douglas et al., 1997; Sugden et al., 1999).The control of the phosphorylation status of specific sites on SPS continues to be an area of considerable interest. With respect to Ser-158 and light/ dark modulation of SPS activity, changes in leaf metabolite pools are thought to play at least some role. Inorganic phosphate (Pi) inhibits the PP2A that dephosphorylates and activates phospho-SPS-158 (Weiner et al., 1992(Weiner et al., , 1993. Thus, the decrease in cytosolic [Pi] that may occur in response to induction of photosynthetic metabolism would increase PP2A activity in vivo and therefore promote SPS activation. As cytosolic [Pi] decreases, the concentration of P-esters would increase proportionally. This could also contribute to SPS activation, as Glc-6-P has been shown to inhibit the phosphorylation/inactivation of SPS in lea...