Purification of ornithine aminotransferase by immunoadsorption.

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.

A comparison of ornithine aminotransferase from human and rat sources.

Ornithine aminotransferase was purified from human liver, rat liver and rat kidney. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a subunit molecular weight of 45,000 in all three cases. Estimations of the native molecular weights of ornithine aminotransferase were determined by Sephadex G-200 chromatography in the presence and absence of 0.1% (w/v) Triton X-100. Human and rat enzymes were tetrameric in the presence of detergent but the rat subunits aggregated further in its absence. Characterisation of ornithine aminotransferase from the two rat sources indicated that they were the same protein. The human and rat enzymes were similar but not identical.