Calcium-binding phosphoprotein from pig brain: Identification as a calcium-dependent regulator of brain cyclic nucleotide phosphodiesterase

Abstract

A homogeneous, acidic, Ca 2+-binding phosphoprotein from pig brain has been identified as the mediator of a Ca 2+-dependent activation of a partially purified pig brain cyclic nucleotide phosphodiesterase. Crude extracts prepared from porcine brain have been resolved into two fractions by column chromatography with ECTEOLA-cellulose. One fraction possesses phosphodiesterase activity which is stimulated from 4- to 12-fold, depending upon the preparation and the assay conditions, by nanogram quantities of a purified Ca 2+-binding phosphoprotein from pig brain at 5 × 10 −5, m Ca 2+. The stimulation is eliminated by ethylene glycol-bis(β-aminoethyl ether)- N,N′-tetraacetate. The second fraction contains an endogenous, Ca 2+-dependent activator of the phosphodiesterase activity. Further purification of the crude activator by hydroyxlapatite chromatography, Sephadex G-75 gel filtration, and acrylamide gel electrophoresis has established that the activator and the purified Ca 2+-binding phosphoprotein are identical with respect to Chromatographic behavior, molecular weight, and electrophoretic migration. Both proteins bound Ca 2+ and were stable to boiling for 5 min. At saturating concentrations of the Ca 2+-dependent regulator, half-maximal stimulation of the phosphodiesterase was observed at a Ca 2+ concentration of 4 × 10 −6, m. Purified preparations of the acidic Ca 2+-binding phosphoprotein from bovine adrenal medulla and testis were also active in stimulating the brain phosphodiesterase. Preparations of other known Ca 2+-binding proteins, such as rabbit muscle troponin and pig brain S-100 protein, were nonstimulatory.