Influence of the alternative respiratory pathway on the adenylate pools in heterotrophic Euglena gracilis

Abstract

Etiolated Euglena gracilis Pringsheim, strain Z, were cultured in a lactate medium either in the presence of 2 μM antimycin A for cells adapted to this inhibitor, or in the absence of antimycin A for controls. The adenylates (ATP, ADP and AMP) and the energy charge (EC) were followed during the growth of both types of cells. The effects of KCN, salicylhydroxamic acid (SHAM) and rotenone on the respiration and the adenylate pool, were investigated during the exponental and stationary phases. EC values of controls and antimycin‐adapted cells were not significantly different during culture. In the logarithmic phase, EC of controls was unaffected by 3 mM SHAM, an inhibitor of the alternative pathway, but markedly decreased by 0.3 mM KCN, which inhibits the cytochrome pathway. In contrast, in antimycin‐adapted Euglena, in which the cytochrome pathway was blocked, ATP content and EC were markedly lowered in the presence of SHAM but slightly increased by 0.3 mM KCN. The combination of the preceeding treatments, as well as 15 mM KCN alone, were deleterious for both types of cells, in the logarithmic and the late stationary phases. The data indicate that the energy level in Euglena was dependent on the alternative pathway when the cytochrome pathway was blocked. Such dependence could be explained by the engagement of the first rotenone‐sensitive site of phosphorylation. Indeed, 50 μM rotenone caused a similar relative decrease of oxygen consumption and ATP content in controls and in antimycin‐adapted Euglena. In the absence of cytochrome respiration, the alternative pathway allowed electrons to flow through this first segment of the respiratory chain, and ATP production by the first site of phosphorylation.

Use of a microbial model for the determination of drug effects on cell metabolism and energetics: Study of citrulline‐malate

“…The standard errors calculated on the basis of triplicate experiments were f 10 per cent. 29 Another type of measurement indicated the capacity of the cells to synthesize ATP, in the 30 s period immediately following treatment by a detergent, which rendered the cells permeable to nucleotides and the added ADP and P04-3. This method used a Nucleotimeter 107 (CLV-Interbio), set on 30s ofpreselection and 'peak condition mode', using 100 pl of luciferin-luciferase mixture from firefly lanterns (4L Biosys reactive) dissolved in water and injected into a mixture of 100 pl of cell suspension in Trizma base buffer 50 mM, pH 7.2, containing Cells from L, L + M, L + C, and L + CM were sampled when the cell titres reached 14-1.5 lo5; they were concentrated to 1 x lo7 per ml in the culture medium and then incubated for 2 h at 27" in the presence of either lactate alone (L), malate alone (M), citrulline alone (C) or citrulline malate (CM).…”