Studies on nitrate reductase in soybean callus tissue



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Nitrate reductase activity was detected in soybean (Glycine soja) callus tissue. Enzyme activity was measured by determining the NO2 accumulation using the standard colorimetric technique (sulfanilamide- HC1, N-l-Naphthylethylene-diamine Dihyrochloride). The reaction components of the enzymatic assay system were tested for their individual effects on the enzyme activity and on the color formation per se. The optimal reaction concentrations of KNO3 and NADH were 8-10 umoles and 0.1 umoles respectively. At concentrations greater than 0.1 umoles, NADH interferred with not only the color formation but also enzyme activity. At the high concentrations of 50 and 100 pinoles, KNO3 inhibited activity. The activity was also lower in tris-HCl buffer than in phosphate buffer. Concentrations of cysteine and EDTA (2 and 10 mM, respectively) in the buffers were detrimental to nitrate reductase activity in the crude and (NH4)2S04 purified extracts, but not so in PEG purified extracts. Nitrite also affected the nitrate reductase reaction. This effect was unexpected since nitrite, itself, is the end product of the enzymatic reaction. The addition of 20 nmoles of NO2 to the reaction mixture prior to incubation enhanced the apparent enzyme activity two to four fold in both crude and partially purified extracts. Certain other nitro-compounds added to the reaction mixture displayed effects similar to those of NO2. It also was determined that blanks in which NADH or KNO3 are omitted from a complete assay may be a source of error in reflecting the true capacity of the tissue for reducing nitrate. Moreover, inactivated (boiled or aged) extracts showed a non-specific accumulation of nitrite not attributable to NR activity, and consequently can not serve as adequate blanks for the NR system.