Cyclic amp-binding proteins and protamine kinases in porcine thyroid cytosol

Abstract

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Iα and Iβ) on DEAE-Sephadex A25. Fractions I, Iα, Iβ comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the following characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [ 3H]AMP showed similar mobilities for Iα, Iβ or II (Rf 0.37) whereas fraction III displayed a much greater mobility ( R F 0.73); Scatchard plots were linear for fractions Iα, II and III with an apparent K d in the same range (2 to 5 nM) whereas fraction Iβ generated a biphasic plot with K d 0.4 nM and 20 nM; cyclic [ 3H]AMP added to fraction I, Iα or Iβ generated a cyclic [ 3H]AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.