Spectrophotometric assay, solubilization and purification of brain 2':3'-cyclic nucleotide 3'-phosphodiesterase.
1. A spectrophotometric assay of 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) based on the use of an acid-base indicator and a buffer having identical pKa values is described. The assay is simple and rapid; it was particularly convenient for monitoring the enzyme activity at various stages of purification. 2. Several proteinases were examined for their ability to solubilize 2':3'-cyclic nucleotide 3'-phosphodiesterase from delipidated brain white matter. Trypsin (EC 3.4.21.4) and elastase (EC 3.4.21.11) appeared to be more effective than the other proteinases examined. Trypsin, however, caused inactivation; elastase was therefore chosen to solubilize 2':3'-cyclic nucleotide 3'-phosphodiesterase. When a partially purified preparation of 2':3'-cyclic nucleotide 3'-phosphodiesterase was treated with elastase, 2':3'-cyclic nucleotide 3'-phosphodiesterase was solubilized nearly quantitatively. Elastatinal, a specific inhibitor of elastase, specifically inhibited the solubilization with elastase. 3. 2':3'-cyclic nucleotide 3'-phosphodiesterase was purified from bovine brain white matter by: (i) delipidation; (ii) solubilization with hexadecyltrimethylammonium bromide; (iii) gel chromatography on Sepharose; (iv) ethanol precipitation and resolubilization by digestion with elastase; (v) chromatography on DEAE-Sephadex; (vi) affinity chromatography on 8-(6-aminohexyl)amino-2'-AMP-Sepharose. 4. The purified enzyme migrated as a single protein band on polyacrylamide-gel electrophoresis at pH 4.3 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the estimated mol.wt. in the latter electrophoresis was 27000-31000. Gel filtration of the purified enzyme through Sephadex G-150 indicated a mol.wt. of 31000. Therefore the purified enzyme is a monomer protein with a mol.wt. of approx. 30000.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D008970:Molecular Weight; D010196:Pancreatic Elastase; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D012995:Solubility; D013053:Spectrophotometry
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D010450:Endopeptidases; D010196:Pancreatic Elastase