PURIFICATION OF 2′,3′‐CYCLIC NUCLEOTIDE 3′‐PHOSPHOHYDROLASE FROM BOVINE BRAIN BY IMMUNOAFFINITY CHROMATOGRAPHY: FURTHER BIOCHEMICAL CHARACTERIZATION OF THE PROTEIN

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Abstract
Abstract— The purification of small amounts of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase from bovine white matter by ion‐exchange techniques (Drummondet al., 1978) has been used to provide antigen for the production of specific rabbit antibodies to this enzyme. Specific antibody has been purified from immune serum by affinity chromatography on a column of Sepharose to which the enzyme has been attached, and the purified antibody has been coupled to cyanogen bromide‐activated Sepharose. Affinity chromatography on the immunoadsorbent effectively purifies 2′,3′‐cyclic nucleotide 3 ‐phosphohydrolase in one step from an extract of an acetone powder made from bovine white matter. This modified purification procedure has reduced the time required for purification and increased the yield of the enzyme to 57%. In SDS‐gel electrophoresis in phosphate buffer the enzyme migrates as an aggregate of about 98,000MW. When the buffer is Tris‐glycine, the apparent MW is about 44,000 and under specific conditions two proteins of only slightly different mobilities can be discerned. Within experimental error the amino acid compositions of the proteins in the two bands are indistinguishable. Peptide patterns obtained by polyacrylamide gel electrophoresis following proteolytic digestion with Straphylococcus aureus V8 protease or papain show extensive structural homology between the two proteins, but detectable differences are apparent.

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