Chick Calretinin: Purification, Composition, and Metal Binding Activity of Native and Recombinant Forms

Abstract

Chick calretinin has been previously expressed inEscherichia coliand purified to homogeneity [Cheung, W-T., Richards, D.E., and Rogers, J.H. (1993)Eur. J. Biochem.215, 401–410]. In the present study we have developed an improved purification procedure, involving a heat precipitation step followed by DEAE–cellulose chromatography with calcium-dependent elution. Native calretinin was purified from chick brainstem using the same method as for the recombinant protein but with an added affinity chromatography step. Typically 30 g of brainstem yielded 350 μg of protein. Several differences between the two forms imply that the native protein is acetylated at the N-terminus but otherwise unmodified. The calcium binding activities of both forms of calretinin were measured by equilibrium dialysis with45Ca in Ca2+/EGTA buffers. The recombinant form bound 4.9 ± 0.12 calcium ions withKd= 0.38 ± 0.02 μmand the native form was not significantly different. Recombinant calretinin was used to study its interaction with other cations present in cells and it was found that calcium binding was affected by Mg2+. Calretinin appears to bind 4.69 ± 0.13 magnesium ions withKd= 4.5 mm. Mg2+increased the apparent dissociation constant for Ca2+. The shift is consistent with competitive binding of Ca2+and Mg2+to the same five sites, but Mg2+binding is too weak to interfere significantly with Ca2+binding under physiological conditions.