Purification by affinity chromatography and preliminary characterization of ornithine decarboxylase from simian virus 40-transformed 3T3 mouse fibroblasts |
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Authors: | Robert J. Boucek Kenneth J. Lembach |
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Affiliation: | 1. Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 USA;2. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 USA |
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Abstract: | Ornithine decarboxylase (l-ornithine carboxy-lyase, EC 4.1.1.17) has been purified from simian virus 40-transformed 3T3 mouse fibroblasts by a procedure utilizing affinity chromatography as the principal step. Selective elution of the enzyme from a pyridoxamine 5′-phosphate-agarose affinity matrix with the use of pyridoxal 5′-phosphate effected a single-step purification of approximately 500-fold, with a significantly higher overall recovery of activity (30 to 45%) than achieved with previous procedures. In the presence of optimal protein concentrations, the enzyme from transformed fibroblasts exhibited a significantly higher specific activity than reported previously for the decarboxylase purified from liver. The apparent affinities of the fibroblast enzyme for substrate and cofactor were similar to those reported for the decarboxylases purified from other tissues. With the use of sodium dodecyl sulfate-gel electrophoresis, the subunit molecular weight of the purified ornithine decarboxylase was demonstrated to be approximately 55,000, while the apparent molecular weight of the active enzyme in vitro as determined by gel filtration was approximately 110,000. |
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