PURIFICATION AND CHARACTERIZATION OF THE RECOMBINANT HUMAN CALCIUM-BINDING S100 PROTEINS CAPL AND CACY

The S100 proteins CAPL and CACY are expressed in a tissue- and cell-sp ecific manner and have been reported to be associated with the metasta tic phenotype of tumor cells. In order to study the biochemical, catio n-binding, and conformational properties, we produced and purified lar ge amounts of the recombinant human proteins in Escherichia coil. Seve ral characteristics of native proteins are shown to correspond to thos e of the bacterially expressed proteins. Both are able to form homodim ers in vitro, probably the biologically active species, but not hetero dimers. The Ca2+-binding parameters were studied by flow dialysis at p hysiological ionic strength. Both isotherms show a maximum of two Ca2 per protein and are insensitive to Mg2+, indicating that the sites ar e of the Ca2+-specific type. The isotherms show slight (CAPL, n(H) 1.1 5) or pronounced (CACY, n(H) = 1.33) positive cooperativity with K-0.5 values of 0.32 mM (CACY) and 0.15 mM (CAPL), indicating that the site s are of the low-affinity type. Conformational changes in the Tyr micr oenvironment of CACY indicate that Ca2+ binding induces a shift of Tyr to a less polar environment. Mg2+ does not affect the fluorescence pr operties nor does it induce a difference spectrum, thus suggesting tha t at physiological ionic conditions it does not interact with the prot ein. The Ca2+-induced difference spectra of CAPL are about 3 times sma ller than those of CACY, suggesting that the additional Tyr84 in CACY is much more sensitive to Ca2+ than the two Tyr residues conserved in both proteins.