Spinach Thylakoids... HELP!

Paul S. Brookes pbrookes at ion.ucl.ac.uk
Sun Jul 19 14:42:50 EST 1998


Hi.  I'm a neurochemist not a "photosynthesist", but am doing some
experiments on spinach thylakoids.  I've prepared them using the method
outlined below, and incubated them in a 4 celcius thermostatted oxygen
electrode inside a dark box.

I get a good light/dark response (slide projector light, ferricyanide as
electron acceptor, uncoupled with CCCP), but after 2-3 minutes of
photosynthesis they stop dead!

None of the following compounds, which I thought might be "rate limiting",
recovered the rate:  NADP+, ADP, Bicarbonate, NH4Cl (to uncouple),
Ascorbate.  Shutting off the light and leaving them in the dark for 10
minutes had no effect either.  The pH of the medium upon cessation of PS
was unchanged at 7.6-7.8

So, why are my thylakoids dying?

Method...
---------
50g young spinach, stalks removed, soaked in distilled water on silver tray
in daylight for 30 min.   150ml grinding medium comprising 330mM sorbitol,
2mM HEPES, 400uM KCl, 40uM EDTA, pH 7.8 with KOH - made to a slush in dry
ice/ethanol bath then added to chopped leaves and homogenised in "polytron"
for 8 seconds.  Resultant slush filtered through 8 layers muslin into 4
centrifuge tubes, spun at 1500g (4500rpm), 30s.  Supernatant disposed,
pellets resuspended in "osmotic shock medium" - 5mM MgCl2, 10mM HEPES, pH
7.8 with KOH.  Spun at 1500g for 5 min.  Supernatant disposed, pellets
resuspended in "resuspension medium" comprising 330mM sorbitol, 5mM HEPES,
10mM KCl, 1mM MgCl2, pH 7.08.  Suspension stored in dark on ice.

Incubations were performed in resuspension medium, with 40ug chlorophyll in
a final volume of 0.5ml.  Potassium ferricyanide at 0.3mM, Uncoupler (CCCP)
at 40uM.

Any suggestions to make my thylakoids survive longer?

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Many thanks in advance
P.S. Brookes.



________________________________________________________
pbrookes at ion.ucl.ac.uk      Department of Neurochemistry
Dr. Paul S. Brookes               Institute of Neurology
Tel (44/0)171 837 3611 x4209   University College London
Fax (44/0)171 833 1016                      Queen Square
http://www.ion.ucl.ac.uk/~pbrookes          WC1N 3BG, UK






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