The role of molybdenum in the synthesis of nitrate reductase in cauliflower ( Brassica oleracea L. var. Botrytis L.) and spinach ( Spinacea oleracea L.)

Abstract

1. 1.The 185W analogue of nitrate reductase was produced in molybdenum-deficient cauliflower ( Brassica oleracea L. var. Botrytis L.) leaves following petiolar uptake of 185W from a molybdenum-free medium. l-Azetidine-2-carboxylic acid in the nutrient solution had no effect on analogue formation, but puromycin decreased by 75% 185W incorporation into protein in the fraction normally associated with nitrate reductase. The analogue co-purified with spinach ( Spinacea oleracea L.) nitrate reductase. 2. 2.An antiserum prepared against partially purified spinach nitrate reductase inhibited enzyme activity. 3. 3.Using the antiserum a method was developed, based on 50% inactivation of a “standard” nitrate reductase preparation, to measure the amount of material in solution cross-reacting as nitrate reductase protein but having no enzyme activity. Crude extracts and solutions resulting after purification with respect to nitrate reductase were obtained from molybdenum-deficient spinach plants grown with nitrate either as the sole nitrogen source of supplemented with ammonium salt. These were assayed for their content of cross-reacting material. 4. 4.The results indicate that approximately 30% of the normal complement of nitrate reductase protein found in extracts of healthy spinach plants was present in extracts of molybdenum-deficient spinach plants either as aponitrate reductase orits immunochemically cross-reacting sub-units. Purification of crude extracts with respect to nitrate reductase resulted in a preferential and substantial loss of cross-reacting material relative to the enzyme. It is suggested that breakdown of the apoprotein is rapid in vivo compared with that of the holoenzyme. Continued net increase of nitrate reductase resulting from metal incorporation into apoprotein is dependent on protein synthesis.