Effects of Glycidate on Carbon Dioxide Fixation with Isolated Spinach Chloroplasts

Abstract

Glycidate (2,3-epoxypropionate) stimulated CO2 fixation in isolated spinach chloroplasts up to 100%. In the presence of glycidate the initial lag phase was abolished and the chloroplasts exported mainly 3-phosphoglycerate instead of dihydroxyacetone phosphate.Glycolate formation was not inhibited by glycidate, which is in agreement with the observation that preactivated ribulose-1,5-bisphosphate oxygenase is not inhibited by this compound. Furthermore, glycidate had no effect on electron transport (NADP reduction) and photophosphorylation, nor was a change in the coupling ratio observed.Zelitch (23) introduced potassium glycidate as a specific inhibitor ofphotorespiration. The illuminated tobacco leafdiscs showed an increased CO2 assimilation (up to 60%1o). Glycidate as a structural analog of GL3 should serve as a potent inhibitor of GL synthesis in an irreversible active-site-directed reaction. A direct effect of glycidate on the RuBP oxygenase was reported by Wildner and Henkel (22). Whereas glycidate blocked significantly (82%) the oxygenase activity, the carboxylase function of the enzyme was not affected. These experiments were carried out with enzyme samples which were not preactivated with bicarbonate. However, recent results by Badger and Lorimer (3) revealed that not only the carboxylase but also the oxygenase is activated in the presence of bicarbonate. Under these conditions glycidate had no inhibitory effect on the oxygenase activity, as also observed by Zelitch (24,25) and Asami and Akazawa (2).Chollet (7) confirmed the glycidate-dependent stimulation of CO2 assimilation with isolated intact spinach chloroplasts. In contrast to Zelitch (23) he observed that GL formation was not impaired by the presence of glycidate.The mechanism for the glycidate stimulation of CO2 fixation thus seemed even more obscure. Investigations concerning the effect of glycidate on electron transport and photophosphorylation showed that the production of ATP and NADPH was not stimulated in isolated chloroplasts (results of this publication). Another explanation of the glycidate effect could be a change of the end product of the carbon assimilation.The aim of this paper was to find the optimal conditions for the ' Photosynthetic CO2 Fixation. The CO2 fixation was determined by H14CO3-incorporation into chloroplasts under red light (RG 610 nm, Schott, Mainz, Germany; 6.7.10O erg cm-2 s-') at 22 C.The reaction mixture and further details of the incubation and assay conditions are given in the legends to the figures.Separation of Fixation Products. At the end of the incubation time 0.2 ml chloroplast suspension was removed and quickly centrifuged. The supernatant was sucked off and 0.15 ml added to 0.075 ml 1 N HCI, the remaining pellet was treated with 0.05 ml 0.33 N HCI. After vigorous shaking, aliquots of 0.01 ml were taken from each fraction, immersed in Aquasol (New England Nuclear) and the "4C incorporation estimated by liquid scintillation counting. Samples of each supernatant and pellet fraction were spotted on...

D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification, functional characterization, and expression

“…Lane 1, bmPHGDH; lane 2, protein standard ladders (Ali et al, 2004;Tabatabaie et al, 2009;Zhao & Winkler, 1996). Due to the lack of the regulatory domain, bmPHGDH activity was not inhibited by serine, the end product of the phosphorylated pathway, similar to that with PHGDH from plants (Ho et al, 1999;Wildner & Larsson, 1979) and animals (Achouri et al, 1997;Jaeken et al, 1996;Walsh & Sallach, 1965). The inhibition of PHGDH activity by serine was reported in E. coli (Sugimoto & Pizer, 1968), and Bacillus subtilis (Saski & Pizer, 1975), which are type I PHGDHs including the domain.…”