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 cytoplasm of the green alga Volvox carteri. Both proteins have a native molecular mass of 90 kDa as determined by gel filtration. cbp2 was purified to apparent electrophoretic homogeneity, having a subunit molecular mass of 42 kDa as determined by SDS/PAGE. The cbp1 preparation contains a 42-kDa and a 44-kDa band. The cAMP-binding activity is not associated with protein kinase activity. Tryptic peptides of cbp2 were sequenced by automated Edman degradation. Two pairs of peptides differ in one amino acid only, thus pointing to the presence of isoforms of cbp2. Both binding proteins differed from the cAMP-specific phosphodiesterases of V. carteri with respect to charge, molecular mass and binding affinity to N6-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 set of modified cAMP derivatives. Both proteins bind cAMP strictly specifically in the anti conformation; position 1 and 6 of the adenine moiety and at least one of the exocyclic O atoms of the ribose cyclic phosphate moiety are essential. 3-Isobutyl-1-methylxanthine is an effective inhibitor of binding but the natural methylxyanthines are not. At present it is not clear whether cbp1 and cbp2 are individual proteins or isoforms of one another.
Feldwisch
O
O
Institut für Biochemie, Universität zu Köln, Germany.
Lammertz
M
M
Hartmann
E
E
Feldwisch
J
J
Palme
K
K
Jastorff
B
B
Jaenicke
L
L
D000595:Amino Acid Sequence; D002352:Carrier Proteins; D000460:Chlorophyta / Q000737:chemistry*; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D000242:Cyclic AMP