Isolation and purification of calcium-binding proteins from bovine neurohypophyses

Abstract

An acidic calcium-binding protein was isolated from the soluble fraction of the homogenate of ox neurohypophyses. The protein has a molecular weight of 35 000 and a subunit weight of 15 000. The purification procedure involved ammonium sulphate fractionation, DEAE-cellulose chromatography and gel filtration on Sephadex G-100 and Sephadex G-50. Conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated it to be a protein distinct from the S-100 protein and the soluble hormone-binding proteins (neurophysins) abundant in the neurohypophysis. This appears to be the only Ca 2+-binding protein in the soluble part of the homogenate, with an apparent K diss for Ca 2+ of 1.1 × 10 −5 M (at 22°C) and a binding capacity of 2 mol of calcium per mol of protein. Two different Ca 2--binding proteins of molecular weights 16 500 and 68 000, respectively, were identified in the sodium-deoxycholate-soluble proteins from an ox neurohypophysial microsome fraction. One of them (the former) has been isolated in high purity by DEAE-cellulose chromatography and gel filtration on Sephadex G-200. This protein binds 4 mol of calcium per mol of protein with an apparent K diss of 1.0 x 10 -5 M (at 22°C). The sodium-deoxycholate-insoluble proteins from the microsomal fraction also have Ca 2+-binding components. The soluble Ca 2+-binding protein has properties similar to and may be identical to Ca 2+-binding proteins which have been isolated from bovine brain and have been demonstrated to be modulators of brain cyclic nucleotide phosphodesterase and of actinomyosin ATPase. It also resembles Ca 2+-binding proteins isolated from bovine adrenals and the electroplax from electrophorus electricus.