Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden |
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Authors: | Jolanta Jerzykiewicz Małgorzata Konieczna Grażyna Kłobus Józef Buczek |
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Institution: | (1) Plant Physiology Department, Institute of Botany, Wrocław University, Kanonia 6/8, 50-238 Wrocław, Poland |
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Abstract: | NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spirodela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolypyrrolidone into the extraction medium had only a slight effect on the stability of the enzyme,
whereas addition of 3 % casein, azocasein, or other proteins to the extraction medium greatly increased the nitrate reductase
(NR) activity. Various protease inhibitors were tested for their ability to prevent the loss of NR activity in vitro. Iodoacetate and para-chloromercuric benzoate, the thiol-protease inhibitors, as well as pepstatin, the aspartic-protease
inhibitor had no effect on stability of the nitrate reductase. EDTA had a slight stimulatory effect, whereas 5 mM o-phenantroline,
another inhibitor of the metallo-proteases increased the activity of nitrate reductase. The highest enzyme activity was found
in the presence of phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-protease inhibitors.
The protease-like inactivator was separated from Spirodela polyrhiza by ammonium sulfate fractionation and acid treatment (pH 4.0). After centrifugation the protein of inactivator in supernatant
adjusted to pH 7.5 was removed. When this fraction was examined by electrophoresis in polyacrylamide which copolymerized with
edestin, the protein of the nitrate reductase inactivator remained at the cathode. Fractions containing a protein of inactivator
degraded casein to products soluble in trichloroacetic acid. Inhibition of the inactivator proteolytic activity by phenylmethylsulphonyl
fluoride and di-isopropyl phosphorofluoridate but not by other reagents (thiol- and metallo-protease inhibitors) suggested
the involvement of a serine residue at its active site. The inactivator fraction from Spirodela polyrhiza resulted in a loss of the nitrate reductase activity in crude extracts from both cucumber and corn seedlings. A biochemical
nature a protein of the nitrate reductase inactivator from S. polyrhiza is discussed. |
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Keywords: | nitrate reductase nitrate reductase inactivator protease inhibitors Spirodela polyrhiza cucumber cotyledons corn roots |
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