COMPARISON OF THE CA2-BINDING PROPERTIES OF HUMAN RECOMBINANT CALRETININ-22K AND CALRETININ()

Calretinin-22k (CR-22k) is a splice product of calretinin (CR) found s pecifically in cancer cells, and possesses four EF-hands and a differe ntly processed C-terminal end, The Ca2+-binding properties of recombin ant human calretinin CR-22k were investigated by flow dialysis and spe ctroscopic methods and compared with those of CR, CR possesses four Ca 2+-binding sites with positive cooperativity (n(H) = 1.3) and a [Ca2+] (0.5) of 1.5 mu M, plus one low affinity site with an intrinsic dissoc iation constant (K'(D)) of 0.5 mM. CR-22k contains three Ca2+-binding sites with n(H) of 1.3 and [Ca2+](0.5) of 1.2 mu M, plus a low affinit y site with K'(D) of 1 mM. All the sites seem to be of the Ca2+-specif ic type. Limited proteolysis and thiol reactivity suggest that that th e C terminus of full-length CR, but not of CR-22k, is in close proximi ty of site I leading to mutual shielding. Circular dichroism (CD) spec tra predict that the content of alpha-helix in CR and CR-22k is simila r and that Ca2+ binding leads to very small changes in the CD spectra of both proteins. The optical properties are very similar for CR-22k a nd CR, even though CR-22k possesses one additional Trp at the C-termin al end, and revealed that the Trp residues are organized into a hydrop hobic core in the metal-free proteins and become even better shielded from the aqueous environment upon binding of Ca2+, The fluorescence of the hydrophobic probe 2-p-toluidinylnaphtalene-6-sulfonate is markedl y enhanced by the two proteins already in the absence of Ca2+ and is f urther increased by binding of Ca2+, The trypsinolysis patterns of CR and CR-22k are markedly dependent on the presence or absence of Ca2+, Together, our data suggest the presence of an allosteric conformationa l unit encompassing sites I-III for CR-22k and I-IV for CR, with a ver y similar conformation and conformational changes for both proteins. I n the allosteric unit of CR, site IV is fully active, whereas in CR-22 k this site has a 80-fold decreased affinity, due to the decreased amp hiphilic properties of the C-terminal helix of this site. Some very sp ecific Ca2+-dependent conformational changes suggest that both CR and CR-22k belong to the ''sensor''-type family of Ca2+-binding proteins.