Grana thylakoids

T) Thylakoids obtained from broker chloroplasts Washed thylakoids resuspended in 5 mM MgCI, and treated with Triton at Triton/Chl]=25 at 4°C ( ) Purified grana-thylakoid membrane fragments separated by centrifugation. 

The RCII complex was extracted with Triton X-100 from spinach grana thylakoids and purified by isoelectric focusing (2), a method which insures a much better stability than with the previous preparations (1). They were kept frozen at 200 K until use. Before measurements, the complex was diluted to an optical density at 673 nm of 2.8 for an optical path of 1 cm in a 50 mM Tris-HCl buffer (pH 7.2) containing 0.22 digi-tonin and 602 (v/v) glycerol. 

Several washings of thylakoids at pH 6.5 and at pH 8.5 release most of the PPase into aqueous solution but the membrane bound PPase activity is also found in the subsequent supernatants, indicating that the thylakoid PPase is rather loosely bound to the outer surface of the membrane. In preliminary experiments we have observed that PPase activity can be seen in both inside out vesicles (grana thylakoids) and right side out vesicles (stroma thylakoids). 

It was further shown that in the stroma-lamellae pLHCP was "free" and thus able to move to the grana thylakoids where it was found in the monomeric and trimeric forms of the pigmented LHCIIb (15). Based on these finding the following model is suggested, for the path taken by pLHCP after it is translocated into the plastid (Fig. 4). 

The nuclear encoded 22 kDa heat shock protein (hsp22) of Chlamydomonas is transported into the chloroplast, where it was localized in the grana thylakoids (9) and apparently protected PSII from photoinhibition (10). In contrast to most other imported proteins, hsp22 was not detected as a high molecular weight precursor in vitro. Therefore it does not have a precursor peptide and seems not to be processed during import in vivo (8). 

Since the hsp22 was recently identified as extrinsic grana thylakoid component of heat shocked Chlamydomonas (9), these results may indicate a different mechanism for hsp22 import, which may be temperature or stress related. 

The protein/chlorophyll ratio of these particles obtained from grana thylakoids is 4.6 +/- 0.4 and corresponds to that of vesicles from spinach [15]. With PSII-membrane preparations oiNicotiana tabacum, which have been obtained according to the method... 

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