title: Isolation and characterization of thylakoid membranes from the filamentous cyanobacterium Nostoc punctiforme.
abstract: Nostoc punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc. A protocol for isolating thylakoid membranes was developed to examine the biochemical and biophysical aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N. punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analysed by measuring oxygen evolution activity, fluorescence spectroscopy and electron paramagnetic resonance spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centres were active in water oxidation in the final membrane preparation. Analysis of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b(6)/f could be observed. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.
authors.affiliation: Department of Photochemistry and Molecular Science, Uppsala University, PO Box 523, SE-75120 Uppsala, Sweden.
authors.forename: Tanai
authors.initials: T
authors.lastname: Cardona
authors.forename: Natalia
authors.initials: N
authors.lastname: Battchikova
authors.forename: Asa
authors.initials: A
authors.lastname: Agervald
authors.forename: Pengpeng
authors.initials: P
authors.lastname: Zhang
authors.forename: Erik
authors.initials: E
authors.lastname: Nagel
authors.forename: Eva-Mari
authors.initials: EM
authors.lastname: Aro
authors.forename: Stenbjörn
authors.initials: S
authors.lastname: Styring
authors.forename: Peter
authors.initials: P
authors.lastname: Lindblad
authors.forename: Ann
authors.initials: A
authors.lastname: Magnuson
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D015180:Electrophoresis, Gel, Two-Dimensional; D046937:Nostoc / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism; D045331:Photosystem I Protein Complex / Q000302:isolation & purification / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000302:isolation & purification / Q000378:metabolism; D020524:Thylakoids / Q000502:physiology
chemical_list: D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D010100:Oxygen