Rapid purification of membrane extrinsic F-1-domain of chloroplast ATP synthase in monodisperse form suitable for 3D-crystallization

A new chromatographic procedure for purification of the membrane extrinsic F-1-domain of chloroplast ATP synthase is presented. The purification is ac hieved by a single anion exchange chromatography step. Determination of the enzyme-bound nucleotides reveals only 1 mole of ADP per complex. The purif ied enzyme shows a latent Ca2+-dependent ATPase activity of 1.0 mu mol.mg(- 1).min(-1) and a Mg2+-dependent activity of 4.4 mu mol.mg(-1).min(-1) Both activities are increased up to 8-10-fold after dithiothreitol activation,An alysis of the purified F-1-complex by SDS/PAGE, silver staining and immunob lotting revealed that the preparation is uncontaminated by fragmented subun its or ribulose- 1,5-bisphosphate carboxylase/oxygenase. Gel filtration exp eriments indicate that the preparation is homogenous and monodisperse. In o rder to determine the solubility minimum of the purified F-1-complex the is oelectric point of the preparation was calculated from pH mapping on ion ex change columns. In agreement with calculations based on the amino acid sequ ence, a slightly acidic pi of 5.7 was found. Using ammonium sulphate as a p recipitant the purified CF1-complex could be crystallized by MicroBatch.