I am not a specialist in plant biochemistry/physiology, but just as a wild
guess, do the plants have carboxysomes? Is that possible that grapevine
carboxysomes are more tough to brake, so that you would require additional
steps in your protocol to lyse them (mild proteolysis of the coat, ...)?
Another wild guess is that for some reason your enzyme may be oxygen
sensitive and you might need to do the whole prep under anaerobic
conditions. Does above make sense? Rubisco is such an abundant enzyme in the
cell that it would be strange not to isolate at least anything. As for
phenols, I guess it would be possible to treat your plant homogenates with
polyphenol oxidases or peroxidases to break down phenol compounds, or to
extract those with organic solvents (cold methanol, acetone...?). Another
thing, can phenols be oxidized under aerobic conditions and coprecipitate
with Rubisco?
Emir
""ITQB"" <jpmaroco at itqb.unl.pt> wrote in message
news:002b01c0d88f$ec59b980$9eb588c1 at itqb.unl.pt...
Hi,
I am trying to extract Rubisco from grapvine leaves, and I generally end up
with none. I use standard extraction buffers (200 mM Tris.Cl pH8.0, MgCl2,
EDTA, etc.) and I have to use up to 10% insoluble PVPP and up to 5% Triton
X100 to actually see some rubisco in SDS-PAGE gels. I believe that the
problem is that Rubisco is binding to phenols and its is being pulled down
during the centrifugation step. Triton does help to solubilize some of this
aggregates, however I do not get consistent results (sometimes works but
most of the time it does not...). I believe it must have to do with plant's
growing conditions (some leaves are more phenols than others). As anybody
run into a similar problem? Any sugestions on how to solubilize Rubisco (in
active form...) from whatever agregates its doing? Thanks a lot.
Joao
**********************************************
João P. Maroco
Lab. Ecofisiologia Molecular - ITQB
Av.Republica. EAN
2780 Oeiras
Portugal
Phone: 214469641
Mobile:965447096
Fax: 214433644
e-mail: jpmaroco at itqb.unl.pt
