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Regulation of Nitrate Reductase Activity in Cultured Spinach Cells as Studied by an Enzyme-Linked Immunosorbent Assay ¹

Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, Japan

An enzyme-linked immunosorbent assay permitting the determination of nanogram quantities of nitrate reductase (NR) in cultured spinach cells has been developed and used for studies of the mechanism by which NR activity is regulated as a function of culture age. When 8-day old spinach cells were transferred to fresh medium, NR activity increased markedly in 2 days and thereafter decreased gradually until it became undetectable on the 10th day after the transfer. Determination of the amounts of NR by the immunosorbent assay indicated that the unique alteration of NR activity could be accounted for by the concomitant change in the amount of NR protein. Immunoblotting analysis of the subunit of NR also supported this result. It is concluded that the regulation of NR in spinach cells as a function of culture age is mediated by changes in the amount of the enzyme protein rather than by activation and inactivation of the preexisting proteins.