Purification and characterization of rat dopamine beta-monooxygenase and monoclonal antibodies to the enzyme.
Dopamine beta-monooxygenase was extensively purified from rat adrenal. The specific activity of the final preparation was approx. 1500 nmol/min per mg protein, which was much higher than the highest yet reported. As judged by gel filtration on Ultrogel AcA22, SDS-polyacrylamide gel electrophoresis, and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 88 000. The isoelectric point of the enzyme was estimated to be pH 6.6 in the presence of 8 M urea. Spleen cells from BALB/c mice immunized with rat dopamine beta-monooxygenase were fused to P3-X63-Ag8-653 mouse myeloma cells. From 55 hybrid cells, 10 stable clones secreting anti-dopamine beta-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4B and the monoclonal antibody-Sepharose was shown to be very useful to isolate rat dopamine beta-monooxygenase from crude preparations.
D000313:Adrenal Medulla / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004299:Dopamine beta-Hydroxylase / Q000276:immunology / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D051381:Rats
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D004299:Dopamine beta-Hydroxylase