Isolation and properties of a cyclic AMP-binding protein from Neurospora. Evidence for its role as the regulatory subunit of cyclic AMP-dependent protein kinase.
A cyclic AMP-binding protein with a native molecular weight calculated to be 82,000 was purified 2,000-fold from Neurospora crassa. The apparent subunit molecular weight was 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native protein exists as a dimer of identically sized subunits. The 8-N3-cyclic [32P]AMP-labeled protein appeared as a doublet upon two-dimensional gel electrophoresis, with pI = 5.4 and 5.5. Binding studies with both noncyclic and cyclic nucleotides showed cyclic AMP to have the highest binding affinity. The KD for cyclic AMP was 300 nM at 23 degrees C. The cyclic AMP-binding protein has two distinct cyclic AMP binding sites, a slowly dissociating site and a rapidly dissociating site. The cyclic AMP analog, 8-bromo cyclic AMP, was found to bind preferentially the slowly dissociating cyclic AMP binding site. The cyclic AMP-binding protein is thought to be the regulatory subunit of cyclic AMP-dependent protein kinase based upon the findings that it co-purifies with this protein kinase activity upon DEAE-cellulose chromatography, isoelectric focusing, and cyclic AMP affinity resin chromatography. On purification by the latter two procedures, no other cyclic AMP-binding activity was detected. A number of similarities between the Neurospora cyclic AMP-binding protein and the cyclic AMP-dependent protein kinase regulatory subunits from yeast and mammalian tissue are discussed.
D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009491:Neurospora / Q000378:metabolism*; D009492:Neurospora crassa / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000302:isolation & purification* / Q000378:metabolism
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D046911:Macromolecular Substances; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D011494:Protein Kinases