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The Ca2+-dependent regulator (CDR) of cyclic nucleotide phosphodiesterase has been purified from the rat testis.The procedure included heat treatment(5 min at 85-90") and conventional column chromatography.Purification recoveries ranged between 55 and 75% with yields of 70 to 130 mg/kg of frozen testes.Rat testis CDR was compared with rabbit skeletal muscle troponin-C, the Ca2+-binding component of troponin.The proteins had nearly identical sedimentation constants (1.9 S), yet would separate during gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The molecular weight has been approximated from sedimentation-diffusion, sodium dodecyl sulfate-gel electrophoresis, and amino acid composition data to be a monomer of 17,000 to 19,000.Some of the physical properties of testis CDR include: a Stokes radius of 23.2 A, a frictional ratio of 1.34, an extinction coefficient (c!ja) of 2.1, and an isoelectric point of pH 3.9.The acidic nature of the protein is reflected in the amino acid composition, in which 35% of the molecule is composed of acidic residues.Testis CDR lacks both cysteine and tryptophan and contains a single histidine and a single trimethyllysine.The NH, terminus is blocked by acetylation and the COOH-terminal amino acid is lysine.Metal binding studies (equilibrium dialysis) demonstrated that testis CDR has four equivalent Ca2+-binding sites with a KD of 2.4 PM.High concentrations of Mg2+ (3 mM) do not compete for these sites.Ca2+ (not Mg2+) produces marked conformational changes in the CDR
Dedman et al. (Thu,) studied this question.