REID, PHILIP D.*, PAUL STEPHENSON, AND DAVID BICKAR. Department of Biological Sciences, Smith College, Northampton, MA 01063, Department of Biology, University of Massachusetts-Amherst, Amherst, MA 01002 and Department of Chemistry, Smith College, Northampton, MA 01063.. - A laboratory exercise for measuring changes in protein content and proteolytic activity during programmed cell death of daylily flowers.
Readily available reagents and simple methods are used to characterize
the dramatic changes in protein levels and protease activity during
floral senescence. Precisely controlled changes in the development of
daylily flowers occurs during a 24h period before and after flower
opening. Students can measure the increase of protein prior to
opening and its subsequent decline by assaying protein extracts from
different time points using the Bradford or CPTS methods. Experiments
using protein synthesis inhibitors such as cycloheximide (CH) can
demonstrate that senescence requires synthesis of new protein and that
the decline in protein is due to the synthesis of proteases and/or
protease activators. Changes in in vivo protease activity can
be visualized by placing petal sections on an agarose gel containing
0.1% gelatin. After removal of the sections, gels stained with
Coomassie Blue reveal clearing where proteases have degraded the
gelatin. Further experiments can be performed with CH treated petals
and different classes of proteases can be characterized by inclusion
of class-specific inhibitors into the agarose/gelatin medium. The
system provides students with the opportunity for a variety of self
designed experiments and for examining the activity of proteases in
other developmental systems. This exercise was also presented at the
1997 ASPP meetings in Vancouver, B.C. Canada
Key words: drop spot, Hemerocallis, protease, protein determination, tissue print