Partial purification of gibberellin oxidases from spinach leaves

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.