地中海拟无枝菌酸菌U-32对硝酸盐的同化及其硝酸还原酶特性

对地中海拟无枝菌酸菌U-32菌株的研究发现,像植物及真菌硝酸还原酶一样,地中海拟无枝菌酸菌U-32硝酸还原酶也是诱导酶,其合成受铵盐阻遏,受硝酸盐的诱导。氯霉素抑制实验的结果表明,该菌株硝酸还原酶的诱导涉及到蛋白质的新合成。钼和钨的竞争实验说明U-32菌株硝酸还原酶也为一钼酶。另外在离体实验中,发现硝酸还原酶活力受到KCN和NADH的抑制,但至今未能找到其生理电子供体。此外,U-32菌株硝酸还原酶也不表现类似于植物的黄递酶等组份酶活性。该菌株硝酸还原酶和其力复霉素产量有一定相关性,但两者确切的关系尚待研究。     

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. 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; )     

A CHLORELLA MUTANT LACKING NITRATE REDUCTASE

Shafer , John , Jr ., James E. Baker and John F. Thompson . (U. S. Plant, Soil and Nutrition Laboratory, U. S. D. A., Ithaca, New York.) A Chlorella mutant lacking nitrate reductase. Amer. Jour. Bot. 48(10): 896–899. 1961.—Following ultraviolet irradiation of Chlorella pyrenoidosa, a mutant was isolated which could not utilize nitrate nitrogen but which could use nitrite, ammonia and some organic nitrogen compounds. This suggested an abnormal nitrate reductase system. Nitrate reductase activity was found in cell-free extracts of wild-type cells but not in similar preparations from the mutant. A test for inhibitor in the mutant extract showed that there was none. Therefore, it was concluded that the mutant lacks an active nitrate reductase.     

RELATIONSHIPS BETWEEN NITRATE REDUCTASE ACTIVITY AND RATES OF GROWTH AND NITRATE INCORPORATION UNDER STEADY-STATE LIGHT OR NITRATE LIMITATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Although activity of the enzyme nitrate reductase (NR) can potentially be used to predict the rate of nitrate incorporation in field assemblages of marine phytoplankton, application of this index has met with little success because the relationship between the two rates is not well established under steady-state conditions. To provide a basis for using NR activity measurements, the relationships among NR activity, growth rate, cell composition, and nitrate incorporation rate were examined in cultures of Thalassiosira pseudonana (Hustedt)Hasle and Heimdal, growing a) under steady-state light limitation, b) during transitions between low and high irradiance (15 or 90 μmol quanta.m^?2.s^?1), and c) under steady-state nitrate limitation. Using a modified assay for NR involving additions of bovine serum albumin to stabilize enzyme activity, NR activity in light-limited cultures was positively and quantitatively related to calculated rates of nitrate incorporation, even in cultures that were apparently starved of selenium. During transitions in irradiance, growth rates acclimated to new conditions within 1 day; through the transition, the relationship between NR activity and nitrate incorporation rate remained quantitative. In nitrate-limited chemostat cultures, NR activity was positively correlated with growth rate and with nitrate incorporation rates, but the relationship was not quantitative. NR activity exceeded nitrate incorporation rates at lower growth rates (<25% of nutrient-replete growth rates), but chemostats operating at such low dilution rates may not represent ecologically relevant conditions for marine diatoms. The strong relationship between NR activity and nitrate incorporation provides support for the idea that NR is rate-limiting for nitrate incorporation or is closely coupled to the rate-limiting step. In an effort to determine a suitable variable for scaling NR activity, relationships between different cell components and growth rate were examined. These relationships differed depending on the limiting factor. For example, under light limitation, cell volume and cell carbon content increased significantly with increased growth rate, while under nitrate limitation cell volume and carbon content decreased as growth rates increased. Despite the differences found between cell composition and growth rate under light and nitrate limitation, the relationships between NR activity scaled to different compositional variables and growth rate did not differ between the limitations. In field situations where cell numbers are not easily determined, scaling NR activity to particulate nitrogen content may be the best alternative. These results establish a strong basis for pursuing NR activity measurements as indices of nitrate incorporation in the field.     

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; )     

REDUCTION OF NITRATE AND NITRITE BY SUBCELLULAR PREPARATIONS OF ANABAENA CYLINDRICA II. REDUCTION OF NITRATE TO NITRITE

Reduction of nitrate to nitrite by particulate preparationsof Anabaena cylindrica was investigated. Preparations whichshowed high activity of nitrate reductase were obtained by sonication(preparation A) or acetone treatment (preparation B). The preparationA also showed a high activity of DPIP-ascorbate photooxidation.The nitrate reductase system accepted electrons from eitherreduced ferredoxin (preparation A & B) or NADH (preparationB), but not directly from NADPH. Ferredoxin was active whenreduced either by action of photochemical system I or by NADPHand NADP-reductase, but dithionite-reduced ferredoxin was completelyinactive. Ferredoxin could be replaced with methyl viologen,benzyl viologen and diquat. Reduced FMN and FAD could serveas electron donors, but the affinity of the reductase towardthese flavin compounds was very low. ¹ This work was supported by grants from the Ministry of Education(4093 and 95612) and from the National Institutes of Health,U.S. (GM-11300).     

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.     

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.     

DISAPPEARANCE OF NITRATE REDUCTASE ACTIVITY FROM CHLAMYDOMONAS REINHARDI

Nitrate reductase activity was induced in Chlamydomonas reinhardi following addition of nitrate. Enzymic activity was assayed in cell-free supernatants and in whole cells whose permeability had been increased by freezing. Nitrate reductase activity in cells decreased rapidly when CO₂-fixation was prevented by (a) darkening cultures, (b) aerating cultures with CO₂-free air, or (c) addition of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). A smaller loss of nitrate reductase activity from darkened cells occurred when (a) acetate-adapted cells were supplied with acetate, or (b) cells were allowed to accumulate carbon reserves by nitrogen starvation before darkening. It was concluded that maintenance of nitrate reductase activity was dependent upon the availability of a suitable carbon source.