Intracellular fate of nitrate reductase in higher plants

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Proteolytic fragments were obtained by limited proteolysis of 120 kDa nitrate reductase from Spinacia oleracea L. using trypsin and Staphylococcus aureus V8 protease. Determination of NH4-terminal sequences in 9 to 14 Edman degradation steps allowed the exact localization of the fragments within the amino-acid sequence of CDNA clone pSPNR117. This clone has a 2324 base insert, and the amino acid sequence deduced from its open reading frame, which contains 640 residues.A sequence identity of 61.2-80.1% was found in the amino acid sequences deduced from the CDNA sequences as obtained by spinach and other higher plant nitrate reductases. However, the amino acid sequences surrounding the proteolytic cleavage sites of nitrate reductase had poor homology.A nitrate reductase-inactivator protein has been purified 16, 000-fold from spinach leaves by PH 5 treatment, chromatography on SE53, Con A-Sepharose, and chromatofocusing. -The yield was 12%, the specific activity was 115 u/mg. Polyacrylamide gel electrophoresis of the final purified inactivator yield 2 major protein bands and both bands exhibited nitrate reductase-inactivator activity. Analysis of this inactivator protein by gel filtration and SDS-gel electrophoresis revealed protein stainable material only in a molecular weight range of 110, 000115, 000. SDS gel electrophoresis under reducing conditions yielded 2 protein bands corresponding to molecular weight of 51, 000 and 53, 00'0- The proteolytic mapping for the two separated subunits appeared similar and possibly identical.