Purification and characterization of rat dopamine β-monooxygenase and monoclonal antibodies to the enzyme |
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Authors: | Sachiko Okuno Hitoshi Fujisawa |
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Institution: | Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-11 Japan |
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Abstract: | Dopamine β-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 β-monooxygenase were fused to P3-X63-Ag8-653 mouse myeloma cells. From 55 hybrid cells, 10 stable clones secreting anti-dopamine β-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 β-monooxygenase from crude preparations. |
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Keywords: | Dopamine β-monooxygenase Monoclonal antibody Catechalomine (Rat adrenal medulla) |
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