Purifications of rat adrenal dopamine-beta-hydroxylase: immunological analysis of its soluble and membrane-bound forms with the use of an antibody raised against the soluble form.
Soluble and membrane-bound dopamine-beta-hydroxylases (sDBH and mDBH, respectively) from rat adrenal glands have been purified through concanavalin A-Sepharose chromatography, gel filtration, and ion-exchange high-performance chromatographies. Both sDBH and mDBH were composed by four subunits of apparent molecular weight of 75,000 and showed a native molecular weight of 300,000. This procedure has not allowed us to obtain a sufficient amount of enzyme to immunize a rabbit. A second, more rapid procedure was designed to isolate sDBH, including concanavalin A-Sepharose chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was raised against this purified protein. The specificity of the antiserum was demonstrated by neutralization of rat adrenal gland DBH activity, labeling of rat adrenal medulla on histological sections, and, after Western blot, labeling of the 75,000-molecular-weight band in the different fractions associated with DBH activity during purification. The antiserum had a higher affinity for the sDBH denatured by sodium dodecyl sulfate than for the native protein. It had a higher affinity for sDBH than for mDBH. These results strongly suggest the presence of specific hydrophilic epitopes on the sDBH, revealing structural differences between the two hydroxylase forms. Two protein bands were stained on Western blots of crude rat adrenal gland extract. One band had an apparent molecular weight of 75,000, and the other of 82,000. Our results showed that the two proteins contained similar epitopes, an observation suggesting a close structural relationship. The higher-molecular-weight protein could be the 75,000 protein before covalent modifications and cleavage.
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D000918:Antibody Specificity; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004299:Dopamine beta-Hydroxylase / Q000276:immunology* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000276:immunology; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D008970:Molecular Weight; D051381:Rats; D012995:Solubility
D007106:Immune Sera; D004299:Dopamine beta-Hydroxylase