Purification of structurally intact grana from plants thylakoids membranes.
Thylakoid membranes in higher plant chloroplasts are composed by two distinct domains: stacked grana and stroma lamellae. We developed a procedure for biochemical isolation of grana membranes using mild detergent to maintain membrane structure. Pigment and polypeptide analyses of membrane preparation showed the preparations were indeed enriched in grana membranes. The method was shown to be effective in four different plant species, although with small changes in detergent concentration. Electron microscopy analyses also showed that the preparation consisted of large membrane patches with roughly round shape and diameter comparable with grana membranes in vivo. Furthermore, protein complexes distribution was shown to be maintained with respect to freeze fracture studies, demonstrating that the protocol was successful in isolating membranes close to their in vivo state.
Morosinotto
Tomas
T
Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58 B, 35121, Padova, Italy. tomas.morosinotto@unipd.it
Segalla
Anna
A
Giacometti
Giorgio M
GM
Bassi
Roberto
R
D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D017360:Arabidopsis / Q000737:chemistry / Q000648:ultrastructure; D002458:Cell Fractionation / Q000379:methods*; D003902:Detergents; D005614:Freeze Fracturing; D001467:Hordeum / Q000737:chemistry / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D045331:Photosystem I Protein Complex / Q000302:isolation & purification; D045332:Photosystem II Protein Complex / Q000302:isolation & purification; D012995:Solubility; D018724:Spinacia oleracea / Q000737:chemistry / Q000648:ultrastructure; D020524:Thylakoids / Q000737:chemistry* / Q000648:ultrastructure*; D003313:Zea mays / Q000737:chemistry / Q000648:ultrastructure
D003902:Detergents; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D000251:Adenosine Triphosphatases