Membrane-bound coupling factor ATPase activity during light-induced chloroplast development in Euglena gracilis

Dark-grown non-dividing cells of Euglena gracilis Klebs var. bacillaris Cori were exposed to light for up to 72 h and thylakoid membrane fractions were isolated by sedimentation in sucrose step gradients at various stages of development. The membrane-bound coupling factor (CF1)-ATPase activity of these prothylakoids (0 h of light) and developing thylakoid membranes (12 to 72 h of light) was characterized by its cation specificity and sensitivity to inhibitors. The enzyme at all stages of development was activated by Mg2+ and to a lesser extent by Ca2+; Mn2+ was found to activate, as well as or better than Mg2 + at comparable concentrations. The activity of the enzyme was almost completely inhibited by dicyclohexylcarbodiimide (DCCD; 0.3 mM), but was insensitive to oligomycin, valinomycin and carbonyl cyanide P-trifluoromethoxyphenylhydrazone (FCCP). Low concentrations of NH4CI gave a slight stimulation of enzyme activity, whereas high concentrations of the uncoupler were inhibitory. The specific activity of the membrane-bound CF,-ATPase was highest in prothylakoid membranes. Specific activity decreased on a thylakoid protein or chlorophyll basis during the first 12 h of development, and achieved a steady state level by 48 h following light induction. Estimates of total CF1-ATPase activity per cell indicate that the time for major synthesis of the enzyme is between 12 and 3d h ol development. These results suggest that following an initial lag period in membrane development lasting about 12 h, there is a formation of CF1-ATPase that accompanies further thylakoid membrane development.