have lab, need enzyme

Dr. David Starrett dstarret at BIOLOGY.SEMO.EDU
Thu Oct 9 09:17:12 EST 1997


>Does anyone know of an enzyme that fits (most of) these criteria?  One in
>which different forms exist in the apoplast and the chloroplast would be
>ideal...leading to simple fractionation experiments.
>

Actually there is one that might work well.  Beta-galatosidase.  There is
easy PNP assay, there are generally 3-4 isozymes expressed in many tissues,
it is hardy (rather resistant to proteases, being left on the bench, etc.),
it has a pH optimum that is within a reasonable range, it shows temperature
dependence, galactose is a wonderful feedback inhibitor, it is
compartmentalized within in the cell, and even cell wall.  I use it from
tomatoes to do all you mention.  In tomatoes, all isozymes appear to be
glycosylated, but at different levels as Con-A can be used to fractionate
the isozymes, to a degree anyways.  Alpha-galactosidase also works, though
there are fewer isozymes.  
  The nice part is extraction is easy: 50 mM NaOAc, pH 6.0 will do.  A
filtering through miracloth, a 10,000 g spin for debris, a 30% Ammonium
Sulfate cut (keep sup) finished up with 80% cut (pellet) yields a good
crude extract.  Dialysis back to extraction buffer is then necesary to get
rid of AS.  Reextracting the filtrate (from miracloth and after a couple of
rinses) can yield cell wall bound isozymes.  This is rough, but
particularly with alpha-gal can show some dramatic differnces.  The
isozymes of B-gal tend to show different pIs so isoelectric focusing can be
used to separate isozymes in some cases.  
  I know it is found in many, many ripening fruits.  Some of the isozymes
are also found in other plant parts.  There are quite a few papers on B-gal
from plants out there, and not all ripening related.
  I am familair with this stuff as it is my research area (and has been for
5 years).  I adapted the extraction of both enzymes to my PP class here.
The students have gotten so into it that half way through the current
semester we have decided to drop some of the demo type experiments and
carry these enzymes through a number of labs.
  Glad to give any more detials, suggestions, etc.
  Good luck,

    Dave Starrett


***********************************************************
* Dr. David Starrett                                      *
* Biology Department, MS 6200                             *
* Southeast Missouri State University                     *
* Cape Girardeau, MO  63701                               *
* Ph: 573-651-2382                                        *
* Fax: 573-651-2223                                       *
* Email: dstarret at biology.semo.edu                        *
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