PURIFICATION OF INACTIVATION FACTOR OF PHOSPHORYLATED NITRATE REDUCTASE FROM LEAVES OF BRASSICA-CAMPESTRIS

The factor that inactivates the phosphorylated nitrate reductase was p urified 870-fold from leaves of Brassica campestris L. ssp. apifera by column chromatography oil DEAE-Toyopearl, Phenyl Sepharose, Sephadex G-200 and Mono Q. Final preparation of the purified factor was nearly homogenous as revealed by polyacrylamide gel electrophoresis and its m olecular weight was estimated to be 70 ku using Superose 12 gel filtra tion column. SDS-polyacrylamide gel analysis of the final preparation showed two protein bands with molecular weight of 30 and 33 ku suggest ing that the inactivation factor was composed of two heterogenous subu nits. The inactivation of phosphorylated nitrate reductase by the puri fied protein was neither rime dependent, nor due to degradation of nit rate reductase, suggesting chat the inactivation factor exhibits its e ffect by binding to the phosphorylated nitrate reductase.