Chick calretinin: purification, composition, and metal binding activity of native and recombinant forms.
Chick calretinin has been previously expressed in Escherichia coli and 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 micrograms 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 with 45Ca in Ca2+/EGTA buffers. The recombinant form bound 4.9 +/- 0.12 calcium ions with Kd = 0.38 +/- 0.02 microM and 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 with Kd = 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.
Stevens
J
J
Physiological Laboratory, University of Cambridge, United Kingdom.
Rogers
J H
JH
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D003001:Cloning, Molecular; D004228:Dithionitrobenzoic Acid; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification; D007525:Isoelectric Focusing; D008274:Magnesium / Q000378:metabolism; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis
D000596:Amino Acids; D064032:Calbindin 2; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D004228:Dithionitrobenzoic Acid; D008274:Magnesium; D002118:Calcium