Abstract
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.