Separation and identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels.
A method is presented for the separation and detection of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels and by immunoblotting. The gel staining procedure is a modification of a method used to demonstrate enzyme activity on blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis. The results show that immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase can be separated under equilibrium conditions on isoelectric focusing gels with an expanded alkaline pH range after solubilization in a mixture of nonionic/zwitterionic detergents and urea. Enzymatically active 2',3'-cyclic nucleotide 3'-phosphodiesterase focused as two closely spaced bands at pIapp 8.1 and 8.8, respectively, while 2',3'-cyclic nucleotide 3'-phosphodiesterase immunoreactivity was detected as four distinct bands at pIapp 4.2, 7.4, 8.8, and 9.3 and a diffuse band at pIapp 7.9-8.2. By two-dimensional separation these five bands showed molecular weights of about 43-47 kDa, i.e., corresponding to reported values for immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase. Since enzyme activity is associated with only two of the bands, nonspecific and artifactual banding due to, e.g., detergent micelle formation, is unlikely.
Persson
H
H
Department of Anatomy, Gothenburg University, Göteborg, Sweden.
Corneliuson
O
O
D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000032:analysis*; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D007525:Isoelectric Focusing; D009185:Myelin Proteins / Q000302:isolation & purification; D010727:Phosphoric Diester Hydrolases; D011817:Rabbits; D013116:Spinal Cord / Q000032:analysis
D015415:Biomarkers; D009185:Myelin Proteins; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase