Purification of rat 2',3'-cyclic nucleotide 3'-phosphodiesterase.
2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37) has been isolated from rat brain myelin by chromatography on successive columns of phenyl-Sepharose CL-4B, CM-Sepharose CL-6B, and 8-(6-aminohexyl) amino-2'AMP-Sepharose 4B. From 15 g of rat brain, approximately 400 micrograms of pure CNP was obtained, with a specific activity of 1,200 (2',3'-cyclic AMP) units/mg protein. The Km of the rat enzyme was 3.7 mM, using 2',3'-cAMP as the substrate. Isoelectric focusing of the enzyme indicated a broad isoelectric range of 8.5-9.0. On SDS polyacrylamide gels, rat CNP appears as two protein bands of approximately 48,000 and 50,000 M.W., with an upper band intensity of about 1/10 that of the lower band. The relative intensities of the bands for CNP and the molecular weights correspond to the Wolfgram proteins W1 and W2 described by other investigators. The amino acid analysis of the purified rat enzyme compared favorably with reported determinations for the bovine enzyme and also with reported values for the rat Wolfgram proteins W1 and W2.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent; D007700:Kinetics; D008970:Molecular Weight; D009186:Myelin Sheath / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D051381:Rats
D000596:Amino Acids; D002413:Cations, Divalent; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases