Purification of a Novel 30, 000 Da Calcium-Binding Protein from Bovine Cerebellum

Abstract

A novel very acidic calcium-binding protein (CaBP) was purified from bovine cerebellum, using ⁴⁵Ca autoradiography as a marker, through a preparative procedure involving salting out with a very high concentration of ammonium sulfate, DE52 column chromatography, RNAase treatment, and HPLC gel filtration. This protein showed a molecular weight of 30, 0000 dalton (Da) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and of 120, 000 on in gel filtration chromatography analysis under physiological ionic strength. The calcium binding activity of this 30, 000 Da CaBP was monitored on the basis of calcium-dependent changes in tyrosine fluorescence (K_d=3.0μM).