calcium-calmodulin measurements

[Nora Plesofsky-Vig]

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  
them. Thanks.

Nora Plesofsky-Vig
nora at biosci.cbs.umn.edu