Highly efficient purificaton of the labile plant enzyme 5-aminolevulinate dehydratase (EC 4.2.1.24) by means of monoclonal antibodies.
5-Aminolevulinate dehydratase (ALAD) from spinach (Spinatia oleracea) was isolated by affinity purification on an immunoabsorbens with a yield of 70 to 80% of the activity in the crude enzyme preparation. The enzyme eluted from the immunoabsorbens was pure as judged by polyacrylamide gel electrophoresis and is a hexamer with a subunit molecular weight of about 50 000. Enzyme bound to the immunoabsorbens was able to synthesize porphobilinogen in a continuous manner. Owing to the lability of the enzyme and its low abundance in plant tissue, we have been unable to obtain similar yields of purified enzyme using classical purification procedures. This highly efficient purification was made possible by using monoclonal antibodies as described by Köhler and Milstein (Nature 256, 495 (1975)). The availability of monoclonal antibodies meant that it was not necessary to purify the enzyme to homogeneity by classical means in order to raise an antiserum specific for ALAD. Sixteen clones of cells producing antibodies against ALAD were selected. They all expressed a chi light chain but differed in the heavy chain class which was eigher gamma 1 or gamma 2a. The availability of pure ALAD enzyme and of highly specific antibodies against the enzyme now enables us to answer questions concerning properties, localization, intercellular transport and evolution of ALAD. It is clear that the technique used and the questions asked are not restricted to ALAD.
D000818:Animals; D000906:Antibodies; D002459:Cell Fusion; D002460:Cell Line; D002846:Chromatography, Affinity; D004355:Drug Stability; D006822:Hybrid Cells / Q000276:immunology; D008214:Lymphocytes / Q000276:immunology; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D010954:Plasmacytoma; D000623:Porphobilinogen Synthase / Q000302:isolation & purification*
D000906:Antibodies; D046911:Macromolecular Substances; D000623:Porphobilinogen Synthase