Rapid purification of membrane extrinsic F1-domain of chloroplast ATP synthase in monodisperse form suitable for 3D-crystallization.
A new chromatographic procedure for purification of the membrane extrinsic F1-domain of chloroplast ATP synthase is presented. The purification is achieved by a single anion exchange chromatography step. Determination of the enzyme-bound nucleotides reveals only 1 mole of ADP per complex. The purified enzyme shows a latent Ca(2+)-dependent ATPase activity of 1.0 mumol.mg-1 min-1 and a Mg(2+)-dependent activity of 4.4 mumol.mg-1 .min-1. Both activities are increased up to 8-10-fold after dithiothreitol activation. Analysis of the purified F1-complex by SDS/PAGE, silver staining and immunoblotting revealed that the preparation is uncontaminated by fragmented subunits or ribulose-1,5-bisphosphate carboxylase/oxygenase. Gel filtration experiments indicate that the preparation is homogenous and monodisperse. In order to determine the solubility minimum of the purified F1-complex the isoelectric point of the preparation was calculated from pH mapping on ion exchange columns. In agreement with calculations based on the amino acid sequence, a slightly acidic pI of 5.7 was found. Using ammonium sulphate as a precipitant the purified CF1-complex could be crystallized by MicroBatch.
Groth
G
G
Heinrich-Heine-Universität Düsseldorf, Biochemie der Pflanzen, Germany. georg.groth@uni-duesseldorf.de
Schirwitz
K
K
D000244:Adenosine Diphosphate / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004229:Dithiothreitol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007526:Isoelectric Point; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry; D008970:Molecular Weight; D009711:Nucleotides / Q000032:analysis; D010940:Plant Proteins / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification*; D018724:Spinacia oleracea / Q000201:enzymology*
D008565:Membrane Proteins; D009711:Nucleotides; D010940:Plant Proteins; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D002118:Calcium; D004229:Dithiothreitol