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 general phenylpropanoid pathway, from Jerusalem artichoke (Helianthus tuberosus L.) (B. Gabriac, D. Werck-Reichhart, H. Teutsch, F. Durst [1991] Arch Biochem Biophys 288: 302-309). Rabbit polyclonal antibodies were raised against the native and denaturated nitrocellulose-bound enzyme. Only the immunoglobulins G (IgGs) elicited upon immunization with native enzyme produced strong inhibition of catalytic activity and good cross-reactivity on western blots. In microsomes from H. tuberosus tissues induced by wounding and various chemicals, a positive correlation between catalytic activity and amounts of immunoreactive protein on western blots was observed. When coupled to cyanogen bromide-activated Sepharose, purified IgGs selectively retained CA4H activity from solubilized plant microsomes. Acid elution from the immunoaffinity matrix provided a rapid procedure for high-yield purification of the CA4H protein. The same IgGs immunoprecipitated a single protein from the in vitro translation products of mRNA isolated from wounded tissues. The apparent molecular weight (57,000) of this polypeptide was identical to that of CA4H purified from tuber microsomes. Immunochemical relatedness between CA4H from different plant species was demonstrated by strong inhibition of catalytic activity and immunopurification of several orthologous enzymes, using IgGs directed against CA4H from H. tuberosus. However, only limited interspecies cross-reactivity was observed on western blots. A careful immunochemical analysis indicates that CA4H immunoreactivity significantly differs from plant to plant. Results are discussed in terms of antibody specificity, enzyme glycosylation, and CA4H regulation.
Werck-Reichhart
D
D
Département d'Enzymologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique UPR 406, Strasbourg, France.
Batard
Y
Y
Kochs
G
G
Lesot
A
A
Durst
F
F
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D006368:Helianthus / Q000201:enzymology*; D007074:Immunoglobulin G; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011817:Rabbits / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology; D050564:Trans-Cinnamate 4-Monooxygenase
D007074:Immunoglobulin G; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase