Purification and Characterization of Nitrite Reductase from Cell Suspension Cultures of Paul's Scarlet Rose and its Cross Reactivity to Antiserum Prepared Against Pea Leaf Nitrite Reductase

Abstract

Nitrite reductase was isolated from nine day old rose cells cultured in NO₃^- and NH₄⁺ medium. The purification procedure involved extraction with Tris-HCl buffer containing EDTA, absorption on DEAE-Cellulose, ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Cellulose and DEAE-Sephadex A-50, gel filtration on Sephacryl S-200 and followed by ferredoxin-Sepharose affinity chromatography. The purified preparation was apparently homogeneous as shown by non-denaturing and denaturing polyacrylamide gel electrophoresis. The sub-unit molecular weight of the purified preparation was found to be 66,000. The K_m value for nitrite was determined to be 0.79 mM. The enzyme activity from rose cells was precipitated by antiserum prepared against nitrite reductase from pea leaves and formed immunoprecipitin bands during immunodiffusion and rocket immunoelectrophoresis.