NITRATE REDUCTASE IN SUGARCANE TISSUES

Nitrate reductase purified from extracts of sugarcane leaf tissuesshowed an absolute requirement for NADH and a partial dependenceon the presence of FAD and Mo⁺⁺⁺. The purified enzyme had poorstability. Activity of nitrate reductase increased toward theyounger nodal regions of the stalk but the enzyme appeared tobe inhibited in the tissues of the apical meristem. Roots showedlow nitrate reductase activity compared to leaf tissue. ¹ Published with the approval of the Director as Paper No. 198in the Journal Series of the Experiment Station, Hawaiian SugarPlanters' Association, Honolulu, Hawaii, U. S. A. This investigationwas supported in part with funds provided by U. S. Departmentof Agriculture (ARS) Contract No. 12-14-100-7788 (34) to theExperiment Station of the Hawaiian Sugar Planters' Association.     

NITRATE REDUCTASE IN PHOTOSYNTHETIC BACTERIUM, RHODOSPIRILLUM RUBRUM. PURIFICATION AND PROPERTIES OF NITRATE REDUCTASE IN NITRATE-ADAPTED CELLS

1. From nitrate-adapted cells of Rhodospirillum rubrum, an activepreparation of nitrate reducing enzyme was isolated in partiallypurified state. The enzyme was found to be localized in thechromatophores of the cell and, on sonication, readily releasedinto the upernatant fraction. The purified enzyme, catalyzingthe electron transfer between DPNH and nitrate, contained ab-type cytochrome, flavin and non-heme iron, which was removedon dialysis in the presence of cyanide. Besides DPNH, only methylviologen(reduced form) was effective as electron donor. 2. The effects of pH and the addition of various activatorsand inhibitors on the rate of nitrate reduction were investigated,using DPNH or reduced methylviologen as the electron donor.The oxidation-reduction of the flavin and the heme in the enzymewas followed spectrophotometrically. A pathway of electron inthe nitrate reduction through this enzyme was proposed. 3. The nitrate reductase of this bacterium was compared withother nitrate reductases obtained from other sources, and themetabolic roles of this enzyme were discussed. In the nitrate-adaptedcells of Rsp. rubrum, only one and the same enzyme was obtainedunder different growth conditions of nitrate assimilation (i.e., nitrate as N-source; light as energy source) and nitrate-respiration(i. e., in the dark; nitrate as hydrogen acceptor and N-source). ¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.This paper was submitted to the University of Tokyo to fulfillthe requirement for the author's doctorate. ² Present address; Botanical Institute, Kyoto University. (Received December 14, 1962; )     

NITRATE REDUCTASE IN PHOTOSYNTHETIC BACTERIUM, RHODOSPIRILLUM RUBRUM. ADAPTIVE FORMATION OF NITRATE REDUCTASE

1. A method was discovered for adapting the cells of Rhodospirillumrubrum to grow on a nitrate medium, a capacity initially lackingin the organism. The adapted cells were able to grow with nitrateas the sole source of nitrogen. The growth responses of theadapted cells towards various nitrogenous sources were investigatedunder various conditions of incubation (aero- and anaerobiosis,light and dark).
2. The adapted cells were found to have simultaneouslyacquiredthe capacity for reducing nitrite and hydroxylamineas wellas nitrate. The path of nitrogen in the adapted cellswas assumedto be as follows: NO₃^– NO₂^– NH₂OH CellularNitrogen.
3. Nitrate metabolism of the adapted cells was investigatedundervarious conditions. In the light, nitrate was reducedand furtherassimilated, leaving insignificant amounts of nitritein themedium. In this case, consumption of nitrate was markedlyinhibitedby other forms of nitrogen (e.g., nitrite, hydroxylamine,aminoacids and ammonium salts). In the dark, nitrate was reducedas the terminal hydrogen acceptor in the oxidative breakdownof organic substances (e.g., malate) in the medium (i.e., nitraterespiration). More nitrite was accumulated in this case thanin the light. Molecular oxygen inhibited the reduction of, aswell as the growth on, nitrate in any of the above cases.
4. Theeffects on the rate of nitrate reduction (and respiratoryoxygenuptake) caused by various experimental factors (pH, nitrateconcentration, electron donors, and addition of hydroxylamine)were investigated, using the resting cells of the adapted organism.

¹ This paper was submitted to the University of Tokyo to fulfillthe requirement for the author's doctorate. ² Present Address: Botanical Institute, Kyoto University, Sakyo-ku,Kyoto. (Received February 14, 1963; )     

GENETIC STUDIES OF NITRATE ASSIMILATION IN ASPERGILLUS NIDULANS

(1) In Aspergillus nidulans, at least 16 genes can mutate to affect the reduction of nitrate to ammonium, a process requiring two enzymes, nitrate reductase and nitrite reductase. (2) niaD is the only gene whose effects on enzyme structure are confined to nitrate reductase alone. It specifies a core polypeptide, one or more of which form the basic subunit of nitrate reductase, molecular weight 50000. (3) At least five cnx genes together specify a molybdenum co-factor, necessary for the activity of nitrate reductase, and of xanthine dehydrogenases I and II. The cnxH gene specifies a polypeptide component of this co-factor, and the cnxE and F gene products are involved in co-factor elaboration, The role of the remaining cnx genes is at present unknown. (4) Functional nitrate reductase has a molecular weight of 200000 and is likely to consist of four subunits, together with one or more molecules of the cnx-specified co-factor. (5) The co-factor plays a catalytic role in the aggregation of nitrate-reductase subunits. (6) The niiA gene is the structural gene for nitrite reductase. (7) Other genes affecting nitrate assimilation are either regulatory or bring about their effects indirectly. (8) Of the genes affecting nitrate assimilation, close linkage is found only between the niiA and niaD genes. (9) Nitrate and nitrite reductases are subject to control by nitrate induction and ammonium repression. (10) Nitrate induction is mediated by the nirA gene whose product must be active for the niiA and niaD genes to be expressed. Since most niaD mutants produce nitrite reductase constitutively, it is likely that the nirA gene product is normally inactivated by nitrate reductase, but only when the latter is not complexed with nitrate, (11) Ammonium repression is mediated by the areA gene, whose product must be active for the expression of the niiA and niaD genes, and which is inactive in the presence of ammonium. (12) The tamA gene may function similarly to the areA gene, both gene products being necessary for the expression of the niiA and niaD genes. (13) Although the niiA and niiD genes are probably contiguous, they are not likely to be organized into a structure equivalent to a bacterial operon. (14) Whereas the areA and nirA genes regulate the synthesis of nitrate and nitrite reductases, it is probable that at least nitrate reductase is also subject to post-translational control, the presence of active enzyme being correlated with high levels of NADPH. (15) The regulation of the pentose-phosphate pathway, of mannitol-I-phosphate dehydrogenase and of certain activities required for the catabolism of some nitrogen-containing compounds appears to be connected with that of nitrate assimilation. In all cases, it is probable that the nirA gene and nitrate reductase itself are involved.     

VARIABILITY IN NITRATE UPTAKE KINETICS IN THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Short-term (1–9 min) nitrate uptake kinetics were measured in Thalassiosira pseudonana (Hust.) Hasle & Heimdal grown in nitrate-limited, ammonium-limited, and nitrate-sufficient continuous cultures. For all cultures, maximal nitrate uptake rates did not develop until approximately 3 min after nitrate addition; thereafter, nitrate uptake rates remained constant or declined slightly. The K_s and V_max for the nitrate-limited cultures were higher at any growth rate than those for the ammonium-limited or nitrate-sufficient cultures. Thus, much higher nitrate concentrations would be required to saturate nitrate uptake in nitrate-limited Thalassiosira pseudonana than is usually considered necessary. The lack of data for other species grown under a range of environmental conditions makes it difficult to generalize about the effect of preconditioning on nitrate uptake kinetics.     

ADAPTIVE FORMATION OF NITRATE REDUCING SYSTEM IN ANABAENA CYLINDRICA

1. Changes in capacities for reducing nitrate, nitrite and hydroxylaminecaused by provision or depletion of various nitrogen sourceswere investigated with a nitrogen fixing blue-green alga, Anabaenacylindrica, and adaptive nature of these reducing system wasdemonstrated.
2. It was found that, under light-aerobic conditions,nitrate-and nitrite- reducing systems were induced by nitrateor nitritebut not by N₂ ammonia and glutamate. On the otherhand, theactivity of enzymes pertaining to hydroxylamine reductionwasstimulated equally by nitrate, nitrite and N₂. The latteractivitywas suppressed markedly in the presence of ammoniaor glutamate.
3. Adaptive formation of nitrite reducing systemis completelyinhibited by chloramphenicol, a potent inhibitorof proteinsynthesis. No formation was also observed under theanaerobiccondition or in the dark.
4. On the basis of thesefindings, a tentative scheme for pathwaysof nitrate reductionand nitrogen fixation in Analaena cylindricawas proposed.

(Received August 22, 1962; )     

INTRASPECIFIC COMPARISONS OF NITRATE UPTAKE IN THREE MARINE DIATOMS1

Short-term (within 5 min) and long-term (≤2 h) rates of nitrate uptake were determined for the marine diatoms, Nitschiella longissima (Cleve), Skeletonema costatum (Greville) Cleve and Asterionella japonica (Cleve). Pigment levels, cell carbon, nitrogen and cell volume were also determined for cells in the logarithmic and stationary phases of growth. For each species, one clone isolated from oligotrophic coastal water and one clone isolated from eutrophic coastal water were compared. Long-term NO₃^? uptake typically followed saturation kinetics describable by the Michaelis-Menten expression. Under experimental conditions, half-saturation constants ranged from 0.6 to 2.2 μM NO₃^?. Generally, the oligotrophic clones had lower Ks and V_max (on a per cell basis) than their eutrophic counterparts, though this was only statistically significant in one pair of clones. Eutrophic and oligotrophic clones also differed in their short-term response to nutrient addition; oligotrophic clones showed greatest rate of uptake at the lowest nitrate addition while uptake by eutrophic clones increased with increasing nitrate concentration. However, all clones had very similar V_max values expressed on a dry weight basis. Under N-starvation, cellular C and pigment levels (and N to a lesser extent) generally declined more in eutrophic than in oligotrophic clones. While the differences between inshore and offshore clones were not great, the results are consistent with the hypothesis that eutrophic waters support algae which grow faster and are less conservative biochemically than cells in oligotrophic waters.     

NITRATE REDUCTASE MUTANTS IN EUDORINA ELEGANS (CHLOROPHYCEAE)1

Three nitrate reductase mutants were independently isolated and characterized in the colonial alga, Eudorina elegans Ehrenberg. nar-1 is a leaky mutant, deficient in the production of nitrate reductase. nar-2 and nar-3 both lack the ability to produce nitrate reductase. However, nar-2 grows and nar-3 does not grow when hypoxanthine is the sole nitrogen source. The specific activity of the next enzyme, in the pathway, nitrite reductase is increased in nar-3 when compared to wild-type, nar-1 and nar-2.     

VARIABILITY OF NITRATE UPTAKE CAPACITY IN MACROCYSTIS PYRIFERA (LAMINARIALES,PHAEOPHYTA) WITH NITRATE AND LIGHT AVAILABILITY1

The nitrate uptake capacity of mature blade tissue of the giant kelp, Macrocystis pyrifera (L.) C. Ag., was examined as a function of the availability of light and nitrate. Time course measurements indicated that nitrate uptake rate, as measured by the incorporation of ¹⁵N, was significantly increased by N starvation. The response was linear over the first hour of exposure regardless of the N status of the tissue indicating that surge uptake was not responsible for the increase. The Michaelis-Menten parameters V_max and K_s, however, were not significantly changed by either growth nitrate concentration or growth irradiance as a result of high variability among blades. Similarly, the initial slope (α) of the nitrate uptake kinetics curves was unaffected. Concentration of photosynthetic pigments increased in response to increased nitrate availability but not to increased growth irradiance. Time course and pigment data demonstrated that mature blade tissue responds to increased N availability by decreasing its capacity to take up nitrate and by increasing its investment in photosynthetic pigments, perhaps for N storage or enhanced light-harvesting capabilities and the increase in reducing power available for N assimilation. This study provides evidence for a dynamic regulatory system that responds to changes in nitrate availability in an integrated manner.