VILIP and NCS-1, neural-specific, 22-kDa Ca(2+)-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties. Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2+ binding) revealed that both recombinant proteins possess only two active metal-binding sites, which can accommodate either Ca2+ or Mg2+. VILIP binds cations without cooperativity with intrinsic affinity constants K'Ca of 1.0 x 10(6) M-1 and K'Mg of 4.8 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding by shifting the isotherms to higher free Ca2+ concentrations without changing their shape. The competition equation yields a K'Mg, comp value of 180 M-1 for both sites. NCS-1 binds two Mg2+ without cooperativity with K'Mg of 8.3 x 10(4) M-1 and two Ca2+ with very strong positive cooperativity (nH = 1.96). In the absence of Mg2+ the K'Ca1 and K'Ca2 values are 8.9 x 10(4) and 1.4 x 10(8) M-1, respectively, which represent an allosteric increase of 1600-fold. Mg2+ shifts the Ca(2+)-binding isotherms to higher Ca2+ concentrations, yielding a K'Mg, comp value of 800 M-1 for both sites. Thus VILIP and NCS-1 show three remarkable differences in the Ca2+/Mg2+ binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperative way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2+/Ca2+ antagonism is much weaker than in NCS-1. Conformational changes monitored by Trp fluorescence indicate that the metal-free forms already are highly structured. Ca2+ binding promotes a 20-30% increase of fluorescence in both proteins, but whereas the Mg2+ form of VILIP has the same fluorescence properties as the metal-free form, Mg(2+)-saturated NCS-1 has those of the Ca2+ form. Near UV difference spectra confirmed that in VILIP the Mg2+ form is very similar to the metal-free form; in NCS-1 it is different, especially in the Tyr region. NCS-1 possesses one unique Cys-38 in EF-hand site I. Its reactivity (kSH) toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for the Ca(2+)- and Mg(2+)-loaded protein, but kSH is 4-fold higher in metal-free NCS-1. VILIP possesses two additional thiols, one of which is inaccessible to DTNB in the native protein. The reactivity of the two accessible thiols is identical in the metal-free and Mg2+ forms and 5-fold higher than in the Ca2+ form.