| Research Abstract |
For mediators of intracellular Ca^<2+> ([Ca^<2+>]_i) signal to cellular mechanisms in the central nervous system (CNS), ubiquitously distributed entities so far are target of intensive studies, yet CNS-specific mediators remain unassessed. In this context, a new type of Ca^<2+>-binding protein has been studied. When rat brain extracts were analyzed by 2-D PAGE followed by ^<45>Ca^<2+> autoradiography, a major 30-kDa spot undergoing marked Ca^<2+>-induced mobility retardation, was detected among more than fifteen spots absent from kidney and liver extracts. This protein, sharing epitopes with calbindin_<28k>, was tentatively termed cephalocalcin for its exclusive localization in CNS as assessed by immunoblotting. Cephalocalcin was isolated from several calbindin_<28k>-immunoreactive or/and Ca^<2+>-binding proteins with molecular masses of -30 kDa by Ca^<2+>-induced selective elution from a DEAE-Sephacel column. Despite a single narrow protein band upon SDS PAGE, the purified protein was
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separated into at least three species by high resolution native PAGE. The three species, purified by DEAE-Sephacel chromatography, showed high affinity Ca^<2+> binding with Kd values (10^<-7>-10^<-6> M) comparable to [Ca^<2+>]_i, and Ca^<2+>-induced marked conformational changes as probed by intrinsic tryptophan fluorescence. Probing of organic molecule-binding sites with 1-anilino-naphthalene-8-sulfonate (ANS) revealed Ca^<2+>-responsive high affinity site(s). Addition of Zn^<2+> augmented the resonance energy transfer from tryptophan to ANS, indicating binding site(s) distinct from those for Ca^<2+>. Unlike calbindin_<28k> and calmodulin, cephalocalcin possessed reactive thiols, whereas formation of disulfides did not account for the occurrence of the different species. Preliminary results suggested that the N-terminal residue of each species was not present with a free amino group. On the basis of the dynamic molecular features common to the three species, combined with evidence of their differential distribution in rat CNS, cephalocalcin is most likely a cluster of molecular forms generated in vivo. Determination of amino acid sequence has been started to fully assign structural differences among the molecular forms. Extensive screening of natural ligands/target molecules is under way on the working hypothesis that cephalocalcin is a new candidate for Ca^<2+>-signal mediator involved in certain functions specific for CNS. Less
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