MONOSPECIFIC POLYCLONAL ANTIBODIES DIRECTED AGAINST PURIFIED CINNAMATE 4-HYDROXYLASE FROM HELIANTHUS-TUBEROSUS - IMMUNOPURIFICATION, IMMUNOQUANTITATION, AND INTERSPECIES CROSS-REACTIVITY

We recently reported the purification of cinnamic acid 4-hydroxylase ( CA4H), a cytochrome P-450 catalyzing the second reaction of the genera l phenylpropanoid pathway, from Jerusalem artichoke (Helianthus tubero sus L.) (B. Gabriac, D. Werck-Reichhart, H. Teutsch, F. Durst [1991] A rch Biochem Biophys 288: 302-309). Rabbit polyclonal antibodies were r aised against the native and denaturated nitrocellulose-bound enzyme. Only the immunoglobulins G (IgGs) elicited upon immunization with nati ve enzyme produced strong inhibition of catalytic activity and good cr oss-reactivity on western blots. In microsomes from H. tuberosus tissu es induced by wounding and various chemicals, a positive correlation b etween catalytic activity and amounts of immunoreactive protein on wes tern blots was observed. When coupled to cyanogen bromide-activated Se pharose, purified IgGs selectively retained CA4H activity from solubil ized plant microsomes. Acid elution from the immunoaffinity matrix pro vided a rapid procedure for high-yield purification of the CA4H protei n. The same IgGs immunoprecipitated a single protein from the in vitro translation products of mRNA isolated from wounded tissues. The appar ent molecular weight (57,000) of this polypeptide was identical to tha t of CA4H purified from tuber microsomes. Immunochemical relatedness b etween CA4H from different plant species was demonstrated by strong in hibition of catalytic activity and immunopurification of several ortho logous enzymes, using IgGs directed against CA4H from H. tuberosus. Ho wever, only limited interspecies cross-reactivity was observed on west ern blots. A careful immunochemical analysis indicates that CA4H immun oreactivity significantly differs from plant to plant. Results are dis cussed in terms of antibody specificity, enzyme glycosylation, and CA4 H regulation.