Large-scale purification and characterization of calmodulin from ram testis its metal-ion-dependent conformers

Abstract

Calmodulin was isolated in large quantities from ram testis by a simple procedure involving sequentially ammonium sulfate fractionation, heat treatment, anion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-200. Divalent cations (Mg 2+ and/or Ca 2+) were present throughout the purification which was entirely performed in the absence of chelators. The final yield was approx. 90 mg per kg testis. Ram testis calmodulin appears to be essentially identical to the brain homologous protein by the following criteria: ultraviolet absorption spectrum, amino acid composition showing a single residue of ϵ- N-trimethyl lysine, and tryptic peptide maps obtained by high performance liquid chromatography. Turkey gizzard myosin light-chain kinase, the activation of which is extremely specific for calmodulin (Walsh, M.P., Vallet, B., Cavadore, J.C. and Demaille, J.G. (1980) J. Biol. Chem. 255, 335–337), was indeed activated by ram testis calmodulin in the presence of calcium. The isolated protein migrated at different rates upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, depending on the absence or presence of divalent metals which probably induce different conformations. The relative migration rates were Ca 2+ > Mn 2+ > Mg 2+ > EDTA. In the presenceof divalent metals, the observed doublet may be ascribed to the equilibrium between ion-free and ion-saturated forms, which exhibited different Stokes radii, as already suggested (Grab, D.J., Berzins, K., Cohen, R.S. and Siekevitz, P. (1979) J. Biol. Chem. 254, 8690–8696).