Abstract
Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [14C]GA12 to [14C]GA15, [14C]GA24 and [14C]GA25, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [14C]Gibberellin A53 was similarly converted to [14C]GA44, [14C]GA19, [14C]GA17 and small amounts of a fourth product, which was preliminarily identified as [14C]GA20, a C19-gibberellin. All GAs except [14C]GA20 were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et. al, Planta 195, 98–107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower.
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Abbreviations
- BSA:
-
bovine serum albumin
- DEAE:
-
diethylaminoethyl
- DTT:
-
dithiothreitol
- GAn :
-
gibberellin An
- GC-MS:
-
combined gas/chromatography-mass spectrometry
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Many thanks and respect from the author to Prof. Jan E. Graebe (University of Göttingen, Germany), Dr. Peter Hedden and Mr. Dennis Ward (both Long Ashton Research Station, Bristol, UK) for helpful discussions and critical reading of the manuscript, to Dr. Dr. Masaji Koshioka (National Research Institute of Vegetables, Ornamental Plants and Tea, Kusawa, Japan) for assistance during parts of the enzyme purification and to Mrs. Gudrun Bodtke for able technical assistance. Special thanks and respect from the author to Mr. Helge Bruelheide, Mrs. Katharina Schweer and Mrs. Annegrit Berghoff (all University of Göttingen) for critical discussions and support. This work was supported by the Deutsche Forschungsgemeinschaft.
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Lange, T. Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm. Planta 195, 108–115 (1994). https://doi.org/10.1007/BF00206298
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DOI: https://doi.org/10.1007/BF00206298