Purification of 2',3'-cyclic nucleotide 3'-phosphohydrolase from bovine brain by immunoaffinity chromatography: further biochemical characterization of the protein

Drummond, R.J.

Journal of Neurochemistry 33(6): 1143-1150

1979


ISSN/ISBN: 0022-3042
PMID: 233239
DOI: 10.1111/j.1471-4159.1979.tb05257.x
Accession: 068524979

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Abstract
The purification of small amounts of 2',3'-cyclic nucleotide 3'-phosphohydrolase from bovine white matter by ion-exchange techniques (Drummond et al.) was used to provide antigen for the production of specific rabbit antibodies to this enyzme. Specific antibody was purified from immune serum by affinity chromatography on a Sepharose column to which the enzyme was attached. The purified antibody was coupled to cyanogen bromide-activated Sepharose. Affinity chromatography on the immunoadsorbent effectively purifies 2',3'-cyclic nucleotide 3'-phosphohydrolase in 1 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 98,000 MW. When the buffer is Tris-glycine, the apparent MW is about 44,000 and under specific conditions 2 proteins of only slightly different mobilities are discerned. Within experimental error the amino acid compositions of the proteins in the 2 bands are indistinguishable. Peptide patterns obtained by polyacrylamide gel electrophoresis following proteolytic digestion with Staphylococcus aureus V8 protease or papain show extensive structural homology between the 2 proteins, but detectable differences are apparent.