The myelin protein CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase): immunoaffinity purification of CNP from pig and rat brain using a monoclonal antibody and phosphorylation of CNP by cyclic nucleotide-dependent protein kinases.
A monoclonal antibody (MAb-46-1) specifically recognizing a 46 kDa basic protein solubilized from brain membranes was used to prepare an affinity column, which allowed a one-step purification of the 46 kDa protein to homogeneity starting from solubilized cerebellar membranes. MAb-46-1 could also immunoprecipitate the 46 kDa protein from solubilized pig or rat cerebellar membranes. Microsequence analysis of affinity purified 46 kDa protein treated with Lys C demonstrated the identity of the 46 kDa protein as a myelin associated protein, i.e. 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37). The amino acid sequences obtained for the porcine CNP were nearly identical with the known sequences of the bovine and human isoforms but only partially with those of rat and mouse CNP. In SDS PAGE the porcine CNP appeared as a doublet of 44.6 and 45.9 kDa. Both bands of the doublet were equally well recognized by MAb-46-1. Porcine CNP was rapidly and specifically phosphorylated by both protein kinase A and cGMP-dependent protein kinase.
Physiologisch-Chemisches Institut, Universität Tübingen, Germany.
R
R
Stricker
F
F
Lottspeich
G
G
Reiser
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D007120:Immunochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009186:Myelin Sheath / Q000201:enzymology* / Q000276:immunology; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D013045:Species Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases