PURIFICATION AND CHARACTERIZATION OF A CAMP-BINDING PROTEIN OF VOLVOX-CARTERI F NAGARIENSIS IYENGAR

Two cAMP-binding proteins, cbp1 and cbp2, were purified from the cytop lasm of the green alga Volvox carteri. Both proteins have a native mol ecular mass of 90 kDa as determined by gel filtration. cbp2 was purifi ed to apparent electrophoretic homogeneity, having a subunit molecular mass of 42 kDa as determined by SDS/PAGE. The cbp1 preparation contai ns a 42-kDa and a 44-kDa band. The cAMP-binding activity is not associ ated with protein kinase activity. Tryptic peptides of cbp2 were seque nced by automated Edman degradation. Two pairs of peptides differ in o ne amino acid only, thus pointing to the presence of isoforms of cbp2. Both binding proteins differed from the cAMP-specific phosphodiestera ses of V. carteri with respect to charge, molecular mass and binding a ffinity to N-6-cAMP-agarose, Reverse-phase chromatography of the bound ligand revealed that the two binding proteins hydrolyse cAMP to 5'AMP . The binding specificity of purified cbp1 and cbp2 was probed by a se t of modified cAMP derivatives. Both proteins bind cAMP strictly speci fically in the anti conformation; position 1 and 6 of the adenine moie ty and at least one of the exocyclic O atoms of the ribose cyclic phos phate moiety are essential. 3-Isobutyl-1-methylxanthine is an effectiv e inhibitor of binding but the natural methylxyanthines are not. At pr esent it is not clear whether cbp1 and cbp2 are individual proteins or isoforms of one another.