Studies on the Retention of CF1 with or without Induced ATPase Activity by Pyrophosphate Treated Thylakoids and Its Relation to the Regeneration of Photophosphorylation

The reconstitution of chloroplast coupling factor 1 (CF1) into thylakoid membranes was investigated by the fluorescence of the covalently attached label fluorescamine. In contrast to a func­tional regeneration of ATP synthesis, a rebinding of CF1 was observed regardless if the protein was in its native, purified state or had been activated for ATPase activity by heat, dithiothreitol (DTT) or trypsin treatment. The reintegration of CF1 into the thylakoid membrane was estimated to be almost quantitative. Since the label binds covalently and irreversibly to free NH2-groups, its effects on the various functional parameters were investigated. Blocking of NH2 groups leads to inhibition of ATPase activity in isolated CF1 . Labeled factor used in a reconstitution experiment on the other hand, does still result in considerable regeneration of ATP formation. Attaching the label to whole thylakoids leads to a differential effect on phosphorylation, which is inhibited, and the H+-uptake which seems largely unaffected. A differential effect is also observed on coupled and uncoupled electron transport. The results are interpreted as evidence for an involvement of free NH2-groups in the mechanism of photophosphorylation.