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Biosynthesis of copper metalloenzymes in subterranean clover

Delhaize, Emmanuel (1986) Biosynthesis of copper metalloenzymes in subterranean clover. PhD thesis, Murdoch University.

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Subterranean clover (Trifolium subterraneum L. cv. Seaton Park) was grown in solution cultures containing adequate nitrogen both with and without copper. After copper deficiency had developed, Cu2+ was added to some deficient plants and copper content, protein content and activities of three copper met a 11oenzymes, (diamine oxidase DAO; [EC, ascorbate oxidase EEC and .o-diphenol oxidase EEC, were assayed in young and recently matured leaf blades over 11 days during the development of the next three leaves.

Copper deficiency had little effect on protein concentrations but markedly depressed enzyme activities and copper concentrations in al 1 leaf blades assayed. Within four days of adding Cu 2 + to copper-deficient plants, copper concentrations of all the leaf blades increased to adequate values. Enzyme activities only increased to control levels in leaves which had not yet emerged at the time that Cu2+ was added.

The results suggested that active holoenzymes of DAO, ascorbate oxidase and .g-diphenol oxidase can only be synthesized in leaf blades during very early stages of their development.

DAO was purified 135-fold from leaves of subterranean clover and found to consist of three isoenzymes. The native enzyme had a molecular weight of about 150,000 and weights of 80,000. Clover DAO catalysed the oxidation of a wide range of amines and was inhibited by copper ligands and reagents consisted of two subunits both with molecular reactive towards carbonyl groups. Copper was essential for enzyme activity with the apoenzyme being reactivated specifically by copper. The enzyme had a broad pH optimum from pH 7 to 8 and an activation energy of 47 kJ/mol with putrescine as a substrate.

To study DAO biosynthesis further, antibodies were raised against the purified enzyme. The biosynthesis of diamine oxidase in leaf blades of subterranean clover was followed by labelling whole plants with 1 4 co2. A pulse-chase experiment in which DAO was immunoprecipitated with anti-DAO antibodies showed that only leaf primordia and the youngest emerged leaves were able to synthesize the enzyme.

The amount of DAO in young leaves of clover grown with a range of copper treatments was determined by its enzymatic activity and by single radial immunodiffusion against anti-DAO antibodies; both parameters were highly correlated with the copper concentration of the 1eaf. Further, anti-DAO antibodies reacted against apo-DAO prepared in vitro indicating that apo-DAO was absent from copper-deficient leaves. These results suggest that the biosynthesis of DAO in young clover leaves is controlled by the copper concentrations of the leaves.

Paly(A) mRNA, purified from leaf primordia and young emerging leaves of plants grown with either a high copper or low copper supply, was translated in wheat germ and rabbit reticulocyte cell-free systems. No differences between the two copper treatments could be seen in the SDS-PAGE profiles of the translation products after f1uorography. Since anti-DAO antibodies did not detect any DAO synthesized in vitro from either treatment no further conclusions could be drawn about the regulation of DAO biosynthesis by copper.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Environmental and Life Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Loneragan, Jack, Webb, John and Dilworth, Michael
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