Characteristics of the binding of Ca2+ and other divalent metal ions to bovine alpha-lactalbumin.

Abstract

Removal of the tightly bound Ca2+ ion from bovine alpha-lactalbumin (Hiraoka et al. (1980) Biochem. Biophys. Res. Commun. 95, 1098-1104) produces a pronounced conformational change, as indicated by fluorescence and absorbance changes. These changes closely resemble the changes that occur on acid denaturation of the native protein. The binding of ions to apo-alpha-lactalbumin at pH 7.4 has been examined by monitoring fluorescence changes and by direct binding measurements with 45CaCl2. The results indicate the presence of two Ca2+ binding sites on apo-bovine alpha-lactalbumin, a stronger binding site (Ka of 2.7 X 10(6) M-1) and a weaker site (Ka of 3.1 X 10(4) M-1); the fluorescence changes on Ca2+ rebinding correlate with saturation of the stronger site. Mn2+ can also bind to restore a "native" structure but with a lower affinity(Ka of 3.5 X 10(5) M-1). Zn2+ and Co2+ do not produce this change, but Zn2+ (1 mM) greatly inhibits the binding of 45Ca2+ in the direct binding assay and produces a time-dependent displacement of Ca2+ from the native protein to an apo-protein-like conformation. Co2+ does not produce these effects. Studies with metal-depleted galactosyltransferase activated with Zn2+ or Co2+ and apo-alpha-lactalbumin or Ca2+-saturated alpha-lactalbumin show that the Ca2+, Zn2+, and apo-alpha-lactalbumin are all able to bind with galactosyltransferase to produce an active lactose synthase complex.