I've used a lab in cell biology in which we assay for succinate
dehydrogenase activity in mitochondria and microsomes to show that it is
possible to identify organelles by their enzyme activity. I learned this
lab from another professor who used fresh mouse liver as a source of
organelles. For various reasons I prefer not to slaughter mice in an
undergrad lab, so I use cauliflower inflorescence instead. This also
enables me to emphasize that plants too have mitochondria and carry out
respiration, and really are alive (many of the students in cell bio are not
truly convinced of this).
In any case, cauliflower mitochondria usually behave beautifully in the
assay. Microsomes (the small vesicles and membrane stuff left after
spinning down the mitos) however give anomolous results. The assay we use
depends on the reduction of a blue dye to the colorless state by transfer
of electrons from succinate dehydrogenase. The cauliflower microsomes
immediately reduce a portion of the dye, decreasing the blue color. The
dye then recovers slowly, presumably with the help of oxygen. This has not
been a big problem as I can explain that since the change is not sustained
and not dependent on succinate it is not the enzyme. This also encourages
them not to slavishly follow directions but to think about what's going on
and what it means.
However, this semester two groups had immediate complete reduction of the
dye--you could see it turn clear before your eyes, even before they could
get it in the spec. One group never saw recovery--it stayed colorless.
Other groups saw only the usual temporary drop and recovery.
What might be in the microsome fraction that is waiting to reduce the dye,
and why did different groups get very different results? Does anyone have
any ideas. Whatever it is, it does not appear to be sustained.
Dr. Gary Coté
Department of Biology
Radford, VA 24142-6931
email: gcote at runet.eduhttp://www.runet.edu:8800/~gcote/