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Spectrophotometric assay solubilization and purification of brain 2' 3' cyclic nucleotide 3' phospho di esterase ec 3.1.4.37


, : Spectrophotometric assay solubilization and purification of brain 2' 3' cyclic nucleotide 3' phospho di esterase ec 3.1.4.37. Biochemical Journal 191(1): 71-82

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. 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 more effective than the other proteinases examined. Trypsin caused inactivation; elastase was 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. 2':3'-Cyclic nucleotide 3'-phosphodiesterase was purified from bovine brain white matter by: delipidation; solubilization with hexadecyltrimethylammonium bromide; gel chomatography on Sepharose; ethanol precipitation and resolubilization by digestion with elastase; chromatography on DEAE-Sephadex and affinity chromatography on 8-(6-aminohexyl)amino-2'-AMP-Sepharose. The purified enzyme migrated as a single protein band on polyacrylamide-gel electrophoresis at pH 4.3 and on sodium dodecyl sulfate/polyacrylamide-gel electrophoresis; the estimated MW in the latter electrophoresis was 27,000-31,000. Gel filtration of the purified enzyme through Sephadex G-150 indicated a MW of 31,000. The purified enzyme is a monomer protein with a MW of .apprx. 30,000.

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