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All three calcium-binding sites participate in the regulation of obelin luminescence BA Illarionov2; VA Illarionova2; VS Bondar2; ES Vysotski2; John R Blinks*1 (1) Friday Harbor Labs., Univ. of Washington, Friday Harbor, WA 98250, USA; (2) Photobiology Lab., Institute of Biophysics, Krasnoyarsk 660036, Russia,      *(blinks@fhl.washington.edu) All Ca-regulated photoproteins contain 3 Ca-binding sites of the EF-hand type. However, it has been suggested on the basis of luminescence titration measurements that only two of them may be involved in the regulation of aequorin luminescence. Implicit in this conclusion are the assumptions that Ca serves as an indispensable reagent in the luminescent reaction, and does not recycle. An alternative view is that Ca serves a purely regulatory role, acting to increase the rate of a reaction that would proceed more slowly without it. In that case, one would expect all three sites to be filled only when the reaction rate was maximal (probably not the case in the titrations). We studied the effects of disabling (by G-for-E substitution in position 12 of the EF-loop) the 3 Ca-binding sites, separately and in all combinations, on the [Ca2+]-effect relation for recombinant obelin. Disabling any of the sites profoundly altered the response to Ca. The influences of Ca binding to the 3 regulatory sites are roughly equal, and are multiplicative. The substitution essentially eliminated Ca binding at sites I and II (numbering from the N-terminus), but reduced the affinity of site III less profoundly. Thus the triple mutant was still capable of responding weakly to very high [Ca]. (Support: NIH, WA Sea Grant, Russ. Acad. Sci.) [Poster: blinks.johnr.62962]

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