Intracellular location of nitrate reductase and nitrite reductase in spinach and sunflower leaves

Summary Chloroplasts have been isolated from spinach and from sunflower which retain their outer membrane and their stroma protein as determined both by ability to fix CO₂ and evolve O₂ at high rates, and by appearance under the phase contrast microscope. Such chloroplasts contain both nitrate and nitrite reductase activity. However, calculations on the distribution of these enzymes, when compared with the distribution of pyruvate kinase and cytochrome c oxidase activity, demonstrate that the larger part of both nitrate and nitrite reductase is located outside of the chloroplast.Supported in part by the National Research Council of Canada.     

The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans.

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.     

Intracellular localization of nitrate reductase, nitrite reductase, and glutamic Acid dehydrogenase in green leaf tissue

Greenhouse grown seedlings of corn (Zea mays L.) and foxtail (Setaria faberii Herrm.) were used as source material in determining the intracellular localization of nitrate reductase, nitrite reductase, and glutamic acid dehydrogenase, Nonaqueous and aqueous isolation techniques were used to establish that nitrite reductase is localized within the chloroplasts, but that nitrate reductase and glutamic acid dehydrogenase are not. Nonaqueous isolation gives distribution patterns of nitrite reductase which are the same as those observed for NADP-dependent 3-phosphoglyceraldehyde dehydrogenase but which differ drastically from the patterns observed for pyruvic acid kinase. The distribution patterns for nitrate reductase are the same as those of pyruvic acid kinase. The techniques used do not eliminate the possibility that nitrate reductase and pyruvic acid kinase are localized on the external chloroplast membrane.

The data obtained establish that glutamic acid dehydrogenase of green leaves is localized within the mitochondria.

     

Effect of chloramphenicol and cycloheximide on the synthesis of nitrate reductase and nitrite reductase in rice leaves

Synthesis of nitrite reductase in rice leaves was inhibited by both cycloheximide and chloramphenicol. This indicated a cooperative action of 70 S and 80 S ribosomes for its synthesis. Nitrate reductase, however, appeared to be exclusively synthesized on the cytoplasmic ribosomes.     

Inheritance of nitrite reductase and regulation of nitrate reductase, nitrite reductase, and glutamine synthetase isozymes

Banding patterns of nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS) from leaves of diploid barley (Hordeum vulgare), tetraploid wheat (Triticum durum), hexaploid wheat (Triticum aestivum), and tetraploid wild oats (Avena barbata) were compared following starch gel electrophoresis. Two NR isozymes, which appeared to be under different regulatory control, were observed in each of the three species. The activity of the more slowly migrating nitrate reductase isozyme (NR1) was induced by NO3- in green seedlings and cycloheximide inhibited induction. However, the activity of the faster NR isozyme (NR2) was unaffected by addition of KNO3, and it was not affected by treatments of cycloheximide or chloramphenicol. Only a single isozyme of nitrite reductase was detected in surveys of three tetraploid and 18 hexaploid wheat, and 48 barley accessions; however, three isozymes associated with different ecotypes were detected in the wild oats. Inheritance patterns showed that two of the wild oat isozymes were governed by a single Mendelian locus with two codominant alleles; however, no variation was detected for the third isozyme. Treatment of excised barely and wild oat seedlings with cycloheximide and chloramphenicol showed that induction of NiR activity was greatly inhibited by cycloheximide, but only slightly by chloramphenicol. Only a single GS isozyme was detected in extracts of green leaves of wheat, barley, and wild oat seedlings. No electrophoretic variation was observed within or among any of these three species. Thus, this enzyme appears to be the most structurally conserved of the three enzymes.     

Occurrence of nitrite reductase in citrus leaves

Evidence is presented for the presence of nitrite reductasein citrus leaves. The enzyme has a Km for nitrite of 45 mu andis inhibited by cyanide. However, unlike citrus nitrate reductase(l), it is probably not a metalloflavo protein, although itmay be related to iron. In addition to the enzymatic nitrite reduction, non-enzymaticnitrite reduction was present in citrus leaf preparations. Underin vivo assay conditions nitrite reduction in one-month-oldleaves was not inhibited by cyanide, in contrast with three-month-oldleaves in which nitrite reduction was almost completely inhibited.Thus it appears that in very young citrus leaves most of thenitrite reduction is non-enzymatic. ¹ Contribution from The Volcani Center, Agricultural ResearchOrganization, P. O. B. 6, Bet Dagan, Israel. Series 1972.........2256AE. (Received November 28, 1972; )     

Regulation of nitrate and nitrite reduction in barley leaves

Reduction of nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaf sections in the dark and in the light, under aerobic (air and mixtures of O₂ and N₂) or anaerobic (N₂) conditions. Oxygen prevented nitrite accumulation but had no effect on accumulated or infiltrated nitrite. Most of the nitrite accumulated under dark-anaerobic conditions was in the "cytoplasmic" (the cell section between the plasma lemma and the tonoplast) fraction of the tissue. Reduction of nitrate was stimulated by 2, 4-dinitrophenol in tissue under dark-air and by 3-(3', 4'-dichlorophenyl)-l, l-dimethyl urea (DCMU) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) in tissue under all environmental conditions studied. Nitrite accumulated in the light in DCMU-treated tissue under N₂ or under aerobic conditions in the presence of CCCP. On its own, CCCP did not promote accumulation of nitrite in leaf sections under light-air. A model for the reduction of nitrate and nitrite is proposed.     

Synthesis and degradation of nitrite reductase in pea leaves

We have shown in a previous publication (Gupta, Beevers 1983 J Exp Bot 34: 1455-1462) that the level of extractable nitrite reductase activity in pea (Pisum sativum cv Burpeeana) leaves is subject to environmental perturbations. In the current study, we have used rocket immunoelectrophoresis to quantitate the level of nitrite reductase protein in extracts from pea plants subjected to various environmental treatments. The level of nitrite reductase cross-reacting material closely followed the level of assayable nitrite reductase activity. The environmental conditions which enhanced the level of extractable nitrite reductase activity resulted in an increased level of nitrite reductase cross-reacting material in the extracts. In contrast, environmental conditions which resulted in a decrease in the level of extractable nitrite reductase activity produced a decline in cross-reacting material. These results indicate that the environmentally induced modulation of extractable nitrite reductase activity involves alteration of enzyme level and is not mediated by a reversible activation-inactivation of the existing enzyme.