Protozoal Enzyme Problem

Heather Martin hcm at
Fri Feb 17 09:30:08 EST 1995

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   

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