Cyclic nucleotide-binding proteins detected by photoaffinity labeling in nucleus and cytoplasm of bovine liver

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Abstract
A photoaffinity labeling method was used to characterize and compare cyclic nucleotide-binding proteins of bovine liver cytosol with binding proteins of the nucleus. After photoaffinity labeling of cytosol with 8-azido cyclic(c)[32P]AMP, autoradiographs of sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed 2 major labeled proteins of 47,000 and 52,000-55,000 daltons. DEAE-cellulose column-derived fractions suggested that the larger protein was the regulatory subunit of peak II cAMP-dependent protein kinase and the smaller protein was the regulatory subunit of peak I kinase. The smaller protein was largely present as the free regulatory subunit. The 2 binding proteins differed in their ability to bind cGMP. Binding to both proteins was abolished by excess unlabeled cAMP but not by 5''-AMP. Photoaffinity labeling of a 0.14 M salt extract of nuclei and a nonhistone chromosomal protein preparation revealed 2 major binding proteins with the same MW and competition profiles as those of the cytosol. Detergent-washed nuclei gave similar results. Several minor binding proteins were observed in both cytosol and nucleus. One protein (36,000 daltons) was unique to the nucleus and had low affinity for 8-azido cAMP. Photoaffinity labeling with c[3H]GMP revealed a cytosol protein, absent from the nucleus, of 31,000 daltons and the ligand was competed for by both cGMP and 5''-GMP. Apparently, the major specific cAMP-binding proteins of bovine liver are the type I and type II regulatory subunits of cAMP-dependent protein kinase and are present in both nucleus and cytoplasm.