Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3',5'-monophosphate dependent protein kinases.
The heat-stable protein inhibitor (Walsh, D. A., et al. (1971), J. Biol. Chem. 246, 1977--1985) of the cyclic adenosine 3',5'-monophosphate dependent protein kinase has been isolated in pure form from rabbit skeletal muscle after a 430 000-fold purification with a 47% yield. The four-step procedure involves sequentially a heat treatment, batchwise anion and cation exchange, and affinity chromatography on protein kinase catalytic subunit covalently coupled to Sepharose 4B. The inhibitor is an acidic protein (pI = 4.24) of molecular weight 11 300. It contains 98 amino acid residues none of which contains sulfur and only 2 (phenylalanine and tyrosine) are aromatic. The NH2-terminus is blocked. The muscle content is ca. 0.6 mg of inhibitor per L of intracellular water. The inhibitor is tightly bound to the catalytic subunit of protein kinase (Ki congruent to 2 X 10(-9) M) and acts competitively with respect to the protein substrates. Protein kinase recognizes a short stretch of the inhibitor sequence, in which arginyl side chains play a crucial role. A study of various competitive inhibitors of the kinase confirms the importance of guanidino groups and hydrophobic side chains in the specific interaction with the substrate binding site.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry
D000596:Amino Acids; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP; D011494:Protein Kinases