Untersuchungen zur lichtabhängigen Nitritreduktion von Chlorella
Summary1.The dependence of nitrite reduction activity in Chlorella fuscaShihira et Krauss, as measured by uptake of nitrite, on the developmental stage, light intensity and CO2 concentration was investigated.2.There is a quantitative relation between uptake of nitrite-nitrogen and increase of organic cell-nitrogen.3.The kinetics of nitrite reduction in light are linear with time at high CO2 concentrations (3% in air) but biphasic at low CO2 concentrations (0 to 0.03%). In the steady state phase nitrite uptake is stimulated by an increase in the CO2 concentration from 0 to 0.03%. The initial phase and the steady state phase exhibit a different temperature dependence.4.Nitrite uptake in the steady state phase is completely inhibited by iodoacetamide at concentrations which inhibit the photosynthetic CO2 reduction (5×10−5 M). At lower iodoacetamide concentrations (1 to 2×10−5 M) the linear time course of nitrite uptake at high CO2 concentrations is changed in the same manner as it is when the CO2 concentration is lowered.It is concluded that under special conditions (e.g. low CO2 concentration) the rate of steady state nitrite reduction in light is limited by the supply of carbon skeleton (i.e. N acceptors in the synthesis of organic nitrogen compounds) from the photosynthetic CO2 reduction cycle.5.Pre-illumination in the presence of nitrite followed by a dark period depresses the initial phase of nitrite uptake in light at low CO2 concentrations. Pretreatment with nitrite in the dark lowers the nitrite uptake of the cells in the initial phase by exactly the same amount of nitrite which has been taken up during the pre-darkening period.
 It is concluded that there is a correlation between the initial phase of nitrite reduction in light and the respiration-linked nitrite reduction in the dark.
Regulation of assimilatory nitrate reduction at the level of nitrite in Chlorella fusca
Batch cultures of Chlorella fusca excreted nitrite into the medium if gassed with air (0.03% CO2), but they did not if supplied with air containing 5% CO2. After a change from high to low CO2 concentration in the gas stream, nitrite excretion started immediately. After an increase in CO2 concentration to 5%, nitrite uptake started within only 30 min. Changes of in-vitro activities of nitrate reductase, nitrite reductase and glutamine synthetase did not correspond to changes of nitrite concentration in the medium and therefore could not explain these observations. A nitrite-binding site, whose activity corresponded with both nitrite excretion and uptake, was detected at the chloroplast envelope. From these data an additional regulatory step in the assimilatory nitrate-reduction sequence is suggested. This includes an envelopeprotein fraction probably regulating the availability of nitrite within the chloroplast.
Untersuchungen über die Nitratreduktion der GrünalgeAnkistrodesmus braunii in vivo und in vitro
Zusammenfassung1.Phasen maximalen Wachstums und optimaler Aktivität der Nitrat und Nitrit reduzierenden Systeme vonAnkistrodesmus braunii liegen zwischen dem 10. und 14. Tag nach Beginn der Kultur.2.Nitrat- und Nitritreduktion unterliegen einer jahreszeitlichen Rhythmik (Maximum im Juli, Minimum im Oktober).3.Neben spezifischen Enzymen sind an der Gesamtnitrat- und Gesamtnitritreduktion intakter Zellen nichtenzymatisch reduzierende Systeme (unter anderem Ascorbinsäure?) beteiligt, deren Aktivität im sauren ph-Bereich am höchsten ist.4.Die Enzyme der Nitratreduktion können im Rohextrakt erst nach Zugabe von Cofaktoren aktiviert werden.5.Die adaptive und lösliche Nitratreduktase benötigt als Flasvinnucleotid FAD und als H-Donator DPNH.6.Die Spezifität der Nitritreduktase gegenüber DPNH bzw. TPNH und FAD bzw. FMN ist gering. Eventuell sind unbekannte Cofaktoren am Mechanismus dieses konstitutiven Enzyms beteiligt. Es wurde in eine grobpartikuläre dissimilatorische sowie eine feinpartikuläre assimilatorische Fraktion getrennt und die Bedeutung energiereicher Phosphate für den assimilatorischen Teil bestätigt.7.Schwermetall- und SH-Gruppen-Gifte inaktivieren die Nitrat- und die nitritreduktase.8.Die ph-Optima werden mit steigendem Reinheitsgrad der Enzyme vom sauren Bereich zum Neutralpunkt hin verschoben.9.Nitrat- und Nitritreduktase konnten um das 23- bzw. 30fache angereichert werden.10.Die an der intakten Zelle erhaltenen Ergebnisse wurden mit den in vitro-Resultaten verglichen.Summary1.The nitrate and nitrite-reducing systems ofAnkistrodesmus braunii have been studied in vivo and in vitro. The rate of growth and the enzymatic activity of the algae are greatest between the 10th and 14th day of culture. In additiion, the reduction of nitrate and nitrite is subject to seasonal changes of activity (maximum in July, minimum in October).2.The adaptive nitrate reductase ofAnkistrodesmus braunii is a soluble enzyme requiring FAD and DPNH. The constitutive and particle-bound nitrite reductase, on the other hand, shows little specificity in its requirements for cofactors, FAD or FMN and TPNH or DPNH being about equally effective Unknown cofactors may be involved in the action of this enzyme. The nitrate and nitrite reductases are sensitive towards poisons binding heavy metals and active sulfhydryl groups. Increasing purification of both enzyme systems leads to a shift of their pH optima from the acid range towards neutrality.3.The nitrite reductase has been separated into two fractions. A dissimilatory nitrite reductase is associated with larger particles, whereas an assimilatory nitrite reductase requiring high-energy phosphateffor its activity is associated with smaller particles.4.In addition to nitrate and nitrite reductases, non-enzymic systems have been found to be involved in nitrate reduction in intact cells. The activity of these systems (possibly ascorbic acid and other reducing substances) ist strongest in an acidic culture medium