Chloroplast membranes contain firmly bound nucleotides. Their synthesis seems not to be dependent on energy. The amount of labelled firmly bound ATP extracted from membranes after incubation in the light of the presence of 32Pi is only slightly affected by uncouplers such as desapidin and CCCP or energy transfer inhibitors as phlorizin at concentrations where steady state phosphorylation is completely abolished. With Dio-9 or NEM, however, the labelling of firmly bound ATP is lowered to a similar extent as the steady state phosphorylation. These effects can be explained assuming a direct modification of the coupling factor. The results of a two stage incubation experiment using a rapid filtration technique support our earlier hypothesis that the gammaP in the liberated ATP does not origin from the previously built phosphorylated intermediate.
Chromatophores of Rhodospirillum rubrum and spinach chloroplasts contain firmly bound ATP that is rapidly labeled along with ADP in the presence of 32Pi and endogenous nucleotides. The labeling is not entirely dependent on light. In chloroplasts three types of bound ATP can be defined methodologically by their extraction properties: buffer-soluble; acid-soluble; and SDS-soluble or firmly bound ATP. Extensive washing of the chloroplasts does reduce buffer-soluble but not acid-soluble and firmly bound ATP. Buffer-soluble [32P] ATP is almost exclusively gamma labeled while acid-soluble and firmly bound ATP are labeled in the beta and gamma position equally. CCCP, desaspidin, and phlorizin do not inhibit the labeling of firmly bound ATP, whereas the phosphorylation is almost abolished. However, EDTA and NEM pretreatments of the chloroplasts affect both reactions similarly. The postillumination [32P] ATP synthesis with chromatophores can be inhibited by adding ATP to the incubation mixture after illumination if 32Pi is included only during the dark incubation, but is without effect if 32Pi is present only during illumination. On the other hand, ADP added after illumination inhibits post-illumination [32P] ATP formation in both chromatophores and chloroplasts only if 32Pi is present during illumination. The data can be explained by a coupling factor having two sites, as proposed previously on the basis that firmly bound ATP does not transfer its phosphoryl group but seems to drive a synthesis of acid-soluble ATP which incorporates free phosphate.
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