Purification of Novel Calcium-Binding Proteins from Bovine Brain

This chapter describes the purification of two novel bovine brain Ca²⁺- binding proteins: CAB-27 and CAB-48. In addition, the strengths and weaknesses of the Chelex assay as a probe for the measurement of Ca²⁺-binding activity in tissue homogenates are discussed. Purified CAB-27 inhibits phospholipase A₂ activity in a dose-dependent manner. Brain Ca²⁺-binding proteins, such as CAB-48, calregulin, calmodulin, S-100, and parvalbumin, have little effect on phospholipase A₂ activity. The Chelex-100 competitive Ca²⁺-binding assay ensures maximum linearity and sensitivity, and this assay is also used as a probe for the detection of Ca²⁺-binding proteins. The studies demonstrated that the majority of the Ca²⁺-binding activity, detected by the Chelex assay, is not associated with calmodulin activity and is based on a comparison of the apparent M_r of the Ca2⁺-binding activity peaks with those of characterized Ca²⁺-binding proteins. The Chelex-100 is a resin of styrene divinylbenzene with acetate as its functional group. The resin binds a variety of divalent cations including calcium and has been used to determine the stability constants of several cation-binding substances. The amount of Chelex to be used in the assay system should be carefully selected to maximize the sensitivity of this assay in the detection of unknown calcium-binding substances in tissue extracts.