Spectrophotometric assay, solubilization and purification of brain 2':3'-cyclic nucleotide 3'-phosphodiesterase

Biochem J. 1980 Oct 1;191(1):71-82. doi: 10.1042/bj1910071.


1. A spectrophotometric assay of 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 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 and elastase (EC 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.

MeSH terms

  • 2',3'-Cyclic-Nucleotide Phosphodiesterases / isolation & purification*
  • Animals
  • Brain / enzymology*
  • Cattle
  • Chemical Phenomena
  • Chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases
  • Molecular Weight
  • Pancreatic Elastase
  • Phosphoric Diester Hydrolases / isolation & purification*
  • Solubility
  • Spectrophotometry


  • 2',3'-Cyclic-Nucleotide Phosphodiesterases
  • Phosphoric Diester Hydrolases
  • Endopeptidases
  • Pancreatic Elastase