Publications

• Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm.

  Lange T.

  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 t ... >> More

  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. << Less

  Planta 195:108-115(1994) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Partial purification of gibberellin oxidases from spinach leaves.

  Gilmour S.J., Bleecker A.B., Zeevaart J.A.

  Four enzyme activities catalyzing the following oxidative steps in the gibberellin (GA) biosynthetic pathway have been extracted from spinach (Spinacia oleracea L.) leaves after exposure to 8 long days: GA(12) --> GA(53) --> GA(44) --> GA(19) --> GA(20). Two of these, GA(53) oxidase and GA(19) oxi ... >> More

  Four enzyme activities catalyzing the following oxidative steps in the gibberellin (GA) biosynthetic pathway have been extracted from spinach (Spinacia oleracea L.) leaves after exposure to 8 long days: GA(12) --> GA(53) --> GA(44) --> GA(19) --> GA(20). Two of these, GA(53) oxidase and GA(19) oxidase, were separable from the other two, GA(44) oxidase and GA(12) 13-hydroxylase, by anion exchange high performance liquid chromatography (HPLC). Apparent molecular weights of the four enzymes as determined by gel filtration HPLC are: GA(12) 13-hydroxylase, 28,400; GA(53) oxidase, 42,500; GA(44) oxidase, 38,100; GA(19) oxidase, 39,500. GA(44) oxidase was purified approximately 100-fold in 0.3% yield by a combination of ammonium sulfate fractionation, anion exchange HPLC, phenyl-Sepharose chromatography and gel filtration HPLC. << Less

  Plant Physiol 85:87-90(1987) [PubMed] [EuropePMC]