1.The interaction between the protein coupling factor of photophosphorylation and its substrate ADP is studied comparatively by two methods : circular dichroism and chemical isolation of the ligand -enzyme complex.2. Experiments with lJC-labelled ADP indicate that the amount of bound nucleotide is proportional to a variation of the circular dichroism spectrum around 260 nm. When coupling factor is saturated with ADP, two moles of ADP are bound per mole of coupling factor.3. The fixation of ADP on coupling factor is inhibited by the presence, in the incubation medium, of phosphate (Pi), pyrophosphate (PPI) or tripolyphosphate ions. The incubation of coupling factor with [32P]PPi shows that PPi inhibition is due to its fixation on the coupling factor. When coupling factor is saturated, two moles of PPi are bound per mole of coupling factor.Chloroplasts which have lost the soluble protein named coupling factor of photophosphorylation are no longer able to synthesize ATP when illuminated in a medium containing ADP and Pi [i]. This factor has been purified [2,3]; its molecular weight is 325000 [4]. After incubation with it, chloroplasts stripped from the coupling factor recover their ability to synthesize ATP under illumination [2]. These experiments indicate that coupling factor is involved in the last steps of photophosphorylation. On the other hand, isolated coupling factor when treated with dithiothreitol [3,5] or trypsin [Z] catalyses, in vitro, the reverse reaction, namely the hydrolysis of ATP. I n view of these properties of the coupling factor, we found it interesting to elucidate the conditions of interaction between coupling factor, ADP or ATP and Pi. Roy and Moudrianakis [6,7] and Forti et al. [8] made use of radioactive nucleotides [3H]ADP and [14C]ADP for the study of the interaction of isolated coupling factor with its substrates. We found that spectroscopic methods, like absorbance or circular dichroism, have revealed no detectable modification of the spectrum in the absorbance range of the protein (around 280nm). By contrast the CD spectrum of ADP (around 260nm) is very sensitive to the adjunction of coupling factor: the Abbreviations. CD, circular dichroism; A A , natural experimental circular dichroism expressed as an absorbance difference ; Ado-P-CH,-P, adenosine 5' diphosphate in which the oxygen bridge between the two phosphorus atoms is replaced by a methano bridge.signal of free ADP is considerably enhanced when coupling factor is mixed with ADP. I n this paper we shall attempt to determine if the interaction, revealed by CD spectra, of ADP on coupling factor is an actual fixation, and to determine its stoichiometry. We shall compare these results to those obtained by a chemical method, i.e. physical separation of the complex on a chromatography column. MATERIALS AND METHODS Chloroplast IsolationThe coupling factor was extracted from spinach chloroplasts prepared by homogenizing 800 g leaves in a blender with 2400 ml extracting medium (0.4 M sucrose-0.02 M Tris pH 7.8). After filtration throug...

The net carbon incorporation in maize (Zea mays) and tomato (Lycopersicum esculentum) leaves was mainly the result of the carboxylation of ribulose 1,5-diphosphate. (20,28) as in the Calvin-type plants (C3 plants). This proposition is supported by enzymic studies (2,8,9,11,35) and the changes in reservoir sizes of RuDP following a CO2 pressure decrease (14, 28). Conflicting results are reported on the location of RuDP and PEP carboxylases in C4 plants (7-9, 12, 36, 37). Wherever the RuDP and PEP carboxylation may be operating in these C4 plants the general scheme is the following. CO2 is first incorporated in C4-dicarboxylic acid and then into PGA by RuDP carboxylase (3).In both types of plants RuDP carboxylation is a key reaction. Nevertheless the RuDP carboxylase activity in tissue extracts of the C2 plants is apparently inadequate to account for

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The adenine nucleotide binding sites on spinach chloroplast coupling factor 1 have been investigated by measuring the fluorescence changes of the substrate or of the enzyme as a function of the amount of nucleotide bound to the protein.The decrease of the fluorescence of ethenoadenine nucleotide and the quenching of the intrinsic fluorescence of the protein has been studied when these nucleotides bind to the factor. The enhancement of the fluorescence of a probe, 4-nitrobenzofurazan (Nbf), is analyzed when adenine nucleotides bind to the protein. These three spectral changes do not parallel the total amount of nucleotide bound to the coupling factor.We propose that the first molecule of nucleotide which binds to the factor exhibits, after binding, specific properties and induces structural changes of the protein. Thus the first molecule of ethenoadenine nucleotide bound to the factor has a very low fluorescence yield and brings about a quenching of the intrinsic fluorescence of the enzyme. Furthermore the first molecule of adenine nucleotide which binds to the factor induces a conformation change which brings the Nbf amino groups from an hydrophilic to an hydrophobic surrounding and consequently causes an enhancement of the fluorescence of the N-Nbf group.