I am a PhD student studying the ciliate protozoal enzymes responsible for the
degradation of bacterial cell walls in the rumen.
I have been running activity gels ( SDS-PAGE with Micrococcus luteus
incorporated into them as a substrate) using sonicate supernates from
Entodinium caudatum, mixed protozoa, and protozoa-free rumen fluid. The
problem is that I have detected 3 bacterriolytic enzymes, of roughly the same
molecular weights from all three fractions. This is a problem, because the
enzymes are present in the protozoa-free preparation ( i.e the bacterial
fraction). It is impossible to say whether the enzymes are from the protozoa,
or from the bacterial contamination of the protozoa. I have tried to eliminate
as many of the bacteria as possible, by washing the protozoa extensively, and
incubating in an antibiotic cocktail, but there are still the endosymbionts
that exist inside, that are virtually impossible to eliminate. We have
considered a molecular approach to the problem, by sequencing the enzyme and
then probing back into the cell to see where the enzyme comes from, but this
is a bit of a shot in the dark.
I would appreciate any suggestion of a solution.
Heather Martin
