npv at MANI.CBS.UMN.EDU
Thu Mar 30 12:31:58 EST 1995
This is a general question on the validity of an indirect approach to
measuring cytoplasmic calcium in cells in response to a stimulus. If
anyone has experience in this area, I would very much appreciate
hearing from them.
Fluorescent dyes for in vivo calcium measurements do not apparently
work well in filamentous fungi (uptake problems and sequestration).
An alternative might be to provide the cells with radiolabeled
calcium and use the calcium-calmodulin complex as an indirect measure
of cytoplasmic calcium. Presumably, this complex could be identified
on a native gel, or an affinity resin for calmodulin might be used to
isolate/enrich for it. Is the calcium-calmodulin complex tight enough
to survive these manipulations? Another possibility might be to
measure (without radioactive calcium) the stimulation of cyclic
phosphodiesterase by cell extracts, but this seems less
straightforward to me.
A side question concerns the reporter protein aequorin. Is its light
emission (reconstituted with coelenterazine) dependent on pH so that
it would change with a change in intracellular pH?
If you have any thoughts on any of these questions I'd like to hear
nora at biosci.cbs.umn.edu
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