Solubilization of 2',3'-cyclic nucleotide 3'-phosphohydrolase from bovine brain without detergents.
The enzyme in brain that hydrolyzes 2',3'-cyclic nucleotides to the 2'-mononucleotides has been found by several authors to be concentrated in the myelin fraction. To facilitate further study of the enzyme, one of our objectives has been to develop a method of solubilizing the enzyme without the use of detergents. When an acetone powder from brain white matter is homogenized with 1 M guanidinium chloride in 0.2 M buffer at pH 6, 10(-3) M in EDTA and in dithiothreitol, the enzyme is solubilized. If the guanidinium chloride is removed by dialysis in a single step, the enzyme reprecipitates, but if a fractional precipitation is performed by reducing the guanidinium chloride concentration by dilution, the enzyme remains in solution at 0.2 M guanidinium chloride. The precipitates obtained in this fractionation probably contain constituents which, at low salt concentration, formed a part of an insoluble aggregate, since after removal of the pellet the supernatant solution can be dialyzed free of guanidinium chloride without precipitating the enzymic activity. The enzyme thus prepared remains in the supernatant when centrifuged at 108,000 X g for 3 h and can be submitted to (NH4)2SO4 fractionation and chromatography on carboxymethyl-Sephadex and on hydroxylapatite. The enzyme has thereby been purified 200-fold in about 20% yield.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000302:isolation & purification*; D000096:Acetone / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002845:Chromatography; D003902:Detergents; D003956:Dialysis; D004229:Dithiothreitol; D005590:Fractional Precipitation; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D008722:Methods; D009186:Myelin Sheath / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D012995:Solubility
D003902:Detergents; D006146:Guanidines; D000096:Acetone; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D004229:Dithiothreitol