Comparative Study of Photophosphorylation Coupling Factor · Ligand Complex by Circular Dichroism and Chemical Isolation

Abstract

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...

Further Study of Nucleotide‐Binding Site on Chloroplast Coupling Factor 1

“…Previously [5] we determined the number of nucleotides bound to the factor by isolation of the nucleotide complex using gel filtration. Because this complex partly decomposes on the Sephadex column, we have found low values for the number of binding sites.…”

“…However, the two 'tight' sites described by Roy and Moudrianakis [4] and by Girault et al [5] are not equivalent, and Girault et al [5] calculated for them two different association constants of 0.8 pM-' and 0.4 pM-'.…”

“…Two nucleotide-binding sites have been generally reported [3,5]. Cantley and Hammes [6] found two equivalent tight ADP-binding sites and a third weak ADP-binding site.…”

“…Girault et al [5] proposed that the second nucleotide-binding site cannot be occupied before the first one has bound one nucleotide molecule. This hypothesis provides a simple explanation of their experimental results giving the amount of bound nucleotides versus the total nucleotide concentration.…”

Tentoxin‐induced adenine nucleotide exchange with soluble and thylakoid membrane‐bound chloroplast coupling factor 1

“…Membrane-bound chloroplast coupling factor 1 (CF1) Contains 1-3 'tightly bound' adenine nucleotides [1][2][3][4][5][6][7][8][9]. These adenine nucleotides exchange with medium ADP upon energization of the thylakoid membrane [2,4,10,11].…”