Isolation and properties of chloroplast coupling factor from wheat.
1. Wheat chloroplast coupling factor (CF1) was extracted with a modification of the chloroform extraction method of Younis et al. (J. Biol. Chem. 252, 1814--1818, 1977). A one-step purification on an 8--25% sucrose gradient yielded a CF1 which was at least 98% pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 2. Inclusion of proteolysis inhibitors during extraction and purification consistently gave a CF1 containing all five subunits. Selective loss of the sigma and epsilon subunits was observed when proteolysis inhibitors were omitted. 3. Proteolysis inhibitors prevented the release of wheat CF1 from the thylakoid by the low-ionic-strength wash method of Strotmann et al. (Biochim. Biophys. Acta, 314, 202--210, 1973). The enzyme extracted with chloroform and low ionic strength were compared by electrophoresis and no evidence of a difference in molecular weight of any subunit was observed. This suggests that a proteolytic event is not required for release of wheat CF1 by the low-ionic-strength method, even though release is inhibited by proteolysis inhibitors. 4. The gamma subunit of wheat CF1 probably contains at least one internal disulfide bridge, as the electrophoretic mobility of this subunit is lower in the presence of reducing agent than in its absence. 5. Wheat CF1 was viewed by the electron microscope after negative staining. Discrete particles, many appearing hexagonal, were observed at high magnifications. Markham rotational analysis confirmed that the enzyme has sixfold symmetry in at least one of its orientations.
D001952:Bridged-Ring Compounds; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides; D008854:Microscopy, Electron; D011480:Protease Inhibitors / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014908:Triticum / Q000032:analysis*
D001952:Bridged-Ring Compounds; D004220:Disulfides; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D006180:Proton-Translocating ATPases