[31] Practical aspects of the use of aequorin as a calcium indicator: Assay, preparation, microinjection, and interpretation of signals

Mitochondrial free [Ca2+] levels and the permeability transition

“…Moreover, aequorin consumption is not a problem to monitor high [Ca 2+ ] M for at least 10 min, as we have shown before using the same aequorin form targeted to the endoplasmic reticulum [13]. In any case, we should also mention that the comparison made by Pitter et al [21], between their in situ calibration and our previous in vitro calibration [22] was made under different conditions of [Mg 2+ ] and temperature, two factors that considerably modify the Ca 2+ -sensitivity of aequorin [15,23], and using a different combination of chelators to prepare the submicromolar Ca 2+ buffers. Thus, in our opinion, confirmation of the different Ca 2+ -sensitivity in situ and in vitro of native aequorin still awaits making the comparison under the same conditions.…”

New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures

“…A major technical challenge has been to devise satisfactory means for nondestructively measuring intracellular free Ca2+ with good time resolution [for reviews see Kretsinger & Nelson (1976) and Ashley & Campbell (1979)l. The most popular technique has been to use dyes or proteins which change their absorption or luminescence upon binding Ca2+ ions. In the past, these indicators have suffered from several problems (Brown et al, 1977; Thomas, 1979;Moisescu et al, 1975;Blinks et al, 1976Blinks et al, , 1978: ( I ) insufficient selectivity against competing cations, particularly H+ and Mg2+; (2) complex stoichiometries of interaction with Ca2+, for example, 1 Ca2+/2 dyes or several Ca2+/1 protein; (3) inflexibility of aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms.…”

Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin

“…Highly purified natural aequorin was obtained from Dr J.R. Blinks (Mayo Foundation, Rochester, MN) [12], and coelenterazine, 2-(p-bydroxybenzyl)-6-(p-hydroxyphenyl)-3,7-dihydroimidazo-(l,2-alpyrazin-3-one), was synthesized by previously described techniques [131.…”