Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Spatial Distribution of Ca 2+ -Binding Proteins 403
Fig. 15. Indicator of calmodulin localization using FL-calmodulin. The sequence
shows mitotic events from 79–121 min where upon cell cleavage occurs. The fluorescence
of FL-calmodulin (final concentration 3 µM) microinjected into sea urchin eggs
is relatively insensitive to Ca 2+ and target protein binding, but provides information
on localization.
3. Simultaneous use of the Ca 2+ -sensitive (TA-calmodulin) and insensitive
(FL-calmodulin) derivative allow us to distinguish between Ca 2+ activation of
calmodulin and local concentration changes of calmodulin (see Fig. 17).
4. To test that calmodulin activation is required for mitotic transitions Trp peptide
(potent calmodulin inhibitor) (12,13) was injected prior to fertilization. Trp peptide
blocks NEB and if injected postNEB blocks the metaphase-anaphase transition
(17). This further suggests that Ca 2+ -calmodulin-dependent processes are
required for mitotic transitions.
4. Notes
1. The three important factors that must be observed while reacting 5-DTAF with
calmodulin are pH, Ca2+ (divalent cation) concentration, and no increase in
reaction time. If these factors are not observed carefully, the ratio of singly labeled