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Incorporation of radioactive molybdenum into protein during nitrate reductase formation and effect of molybdenum on nitrate reductase and diaphorase activities of spinach (<italic>Spinacea oleracea</italic> L.)
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1971
Year
EngineeringPhotorespirationBotanyPlant BiochemistryChemical BiologyRedox BiologySpinach PlantsBiosynthesisReactive Nitrogen SpecieEnzyme ActivityPhotosynthesisRadioactive MolybdenumNitrate Reductase FormationBiochemistryNitrate ReductaseBiomolecular EngineeringPlant MetabolismBiotechnologyMicrobiologyMetabolismMedicineNitrosative StressPlant Physiology
Spinach plants grown without molybdenum lack nitrate reductase and when plants are deprived of nitrate existing activity is lost. Transfer of molybdenum-deficient plants to a solution containing (NH4)299MoO4) or nitrate-starved plants to NaNO3 solution induced enzyme activity in 24 hr. After purification by selective adsorption, precipitation and disc electrophoresis, the protein from molybdenum-deficient plants given 99Mo showed radioactivity only where nitrate reductase was revealed on the acrylamide gel. Molybdenum was similarly selectively concentrated into the enzyme as a result of induction by nitrate in plants grown with sub-optimal molybdenum supply in order to minimize effects of isotope dilution on measurement of 99Mo incorporation. There was no exchange in vitro between 99Mo and purified active enzyme in the resting state over 18 hr at 4°C, or with functioning enzyme held at room temperature for 24 hr. There was evidence either for possible in vivo exchange of 99Mo andenzyme bound Mo or for slight synthesis of fresh enzyme under conditions of net loss of enzyme in nitrate starved plants. Five NADH2 and two NADPH2 reactive diaphorases which could be separated by electrophoresis were present in extracts. Only one of these having strong NADH2 and weak NADPH2 activity was directly associated with nitrate reductase. The same complex also showed the only benzyl viologen (BV.) reactive nitrate reductase. Nitrate reductase in spinach is therefore considered to be a molybdenum-dependant and molybdenum-containing protein in which NADH2 (with weak NADPH2) and BVelectron donor functions and diaphorase/reductase activities remain closely associated during purification and electrophoresis. The techniques provide a simple means for the production and purification of enzyme containing radioactively labelled Mo applicable to investigations on the structure of the enzyme.