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.

摘要: 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 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the exists as dimer of identically sized subunits. 8-N3-cyclic [32P]AMP-labeled appeared doublet upon two-dimensional pI = 5.4 and 5.5. Binding studies both noncyclic nucleotides showed AMP have highest binding affinity. KD for 300 nM at 23 degrees C. has two distinct sites, slowly dissociating site rapidly site. analog, 8-bromo AMP, found bind preferentially is thought regulatory AMP-dependent kinase based findings it co-purifies this activity DEAE-cellulose chromatography, isoelectric focusing, affinity resin chromatography. On purification latter procedures, no other detected. number similarities between subunits yeast mammalian tissue are discussed.