Light - mediated Activation of NitrateReductase in Synchronous

The mhsm underying the sharp increase in activity of nitrate reductase (EC 1.6.6.1) in Chlorefi vulgars forma terdis (strain 211 8k) during the first hour of the 7 hours/5 hours Light/dark cycle was investigated. Using the method of density labelng and isopycnic centaton, It could be trated that this rapi increase in activity is based on ligt-mediated activation ratber than denovo synthesis of the enzyme. The problematic nature of cycloheximide spechfcity and models of nitrate reductase activation are discussed. The reduction of nitrate to ammonia, requiring a total of eight electrons, is catalyzed during the first two steps by the enzymes nitrate reductase (EC 1.6.6.1) and nitrite reductase (EC 1.6.6.4); both enzymes have been frequently investigated in a variety of organisms. During the cell cycle of Chlorella vulgarisforma tertia (20), it was found that the activities of both enzymes exhibited a 28-fold increase in specific activity during the 1st hr of illumination. The cells grew completely synchronously in a light/dark cycle of 7 hr/5 hr. Such an increase in enzymic activity can be due either to de novo synthesis of the enzyme protein or to activation by light of a protein already present during the dark. For a differentiation between the two possibilities, we used the method of density labeling of the enzyme with subsequent isopycnic sedimentation (8, cf. 10). MATERIALS AND METHODS Chlorella vulgarisforma tertia (strain 211 8k of the algal collection of the Institute of Plant Physiology, University of Gottingen) was cultivated as described by Pirson et al. (16) and Lorenzen (14). The alga grows completely synchronously in a LD1 7/5 and produces eight autospores/cell each cycle. Measurement of enzyme activities and preparation of cell-free extract were previously described (20). Protein was estimated by the Lowry method (15). Density labeling was achieved by culturing the algae in the presence of deuterated amino acids (4 mM, 98% deuterated, obtained from Merck, Sharp & Dohme of Canada). Separation was done in both metrizamide (for nitrate reductase) and rubidium chloride (for glutamate dehydrogenase, acid phosphatase) according to Huttermann and Wendlberger (13). Density calculation followed the measurement of refractive indices using the formula: p" = 3.0534 i-2.9541 for metrizamide-2H20 solutions. ' Abbreviation: LD: light/dark changes. RESULTS Conditions of Density Labling. The usual way of performing density labeling studies is to apply the label of heavy amino acids together with the change in physiological conditions which are to be studied (12). This strategy could not be used during the course of this study because introductory experiments showed that even a small amount of external amino acids completely suppressed the increase in nitrate reductase activity after onset of the light. In addition, it was found that supplementing the growth medium with amino acids (4 mM) did not result in an increase in the amino acid pool of the organism (data not shown). Therefore the labeling scheme had to be reversed. The cells used in our experiments had to be pregrown in the presence of deuterated amino acids, then transferred to the standard medium and illuminated without heavy label. Inasmuch as the activity increase of nitrate reductase was repressed by any external amino acids, the heavy amino acids should be depleted from the pool before the expected synthesis of nitrate reductase. Any protein being synthesized before illumination thus should contain deuterated amino acids, later produced proteins should be composed mainly by light amino acids. Intermal Markers. As intemal density markers and indicators for over-all protein synthesis, NAD-glutamate dehydrogenase (EC 1.4.1.2) and acid phosphatase (EC 3.1.3.2) were chosen which both could be found as single bands in the density gradients. Furthermore, these specific activities behave in a way considerably different from those of nitrate reductase (Table I). Assuming the 1st hr of the light cycle, where nitrate reductase exhibits an increase in activity of 2,850%, both glutamate dehydrogenase and acid phosphatase decrease slightly in their specific activities. The same behavior was observed in the presence of actidion, whereas the nitrate reductase was inhibited in its activity of 98%. The residual enzyme activity was much higher in the case of the two other enzymes. These differences in the behavior of the specific activities, 28.5-fold increase in the case of nitrate reductase, and a slight decrease in the two other enzymes, should be measurable by density labeling experiments, if actual de novo synthesis of the respective enzymes is involved. Density Labeling Experiments. In all density labeling experiments the cells were grown in the presence of deuterated amino acids (4 mM) for three generations in order to reach equilibrium labeling. For the induction experiments the cells were harvested at the end of the last dark period, washed twice with the standard nutrient solution, resuspended in the same solution, and illuminated for 1 hr. A parallel culture grown in the absence of deuterated amino acids and handled in the same way was used as reference. Aliquots of the extracts were centrifuged both in metrizamide-2H20 (for nitrate reductase) and RbCl-H20 (for glutamate dehydrogenase and acid phosphatase) density gradients. The fractions were then analyzed for appropriate enzyme activity. Initial 284 www.plantph siol.org on July 26, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. LIGHT ACTIVATION OF NITRATE REDUCTASE studies had shown that nitrate reductase is unstable in the presence of CsCl or RbCl. Therefore, metrizamide-2H20 gradients had to be used for its isopycnic banding. Unfortunately, the capacity of that gradient was too small to permit assay of the activities of all three enzymes. So parallel samples of the same extract were centrifuged in RbCl gradients. The result of such an experiment is given in Figure 1. Nitrate reductase of cells pregrown with heavy amino acids bands at a density ofp2 equals 1.2422, whereas the enzyme from algae grown in the absence ofamino acids is significantly lighter (p25 = 1.2300). Mixed gradients made with both extracts show a double peak. Identical results were found for the two internal markers. Figure 2 shows the results only for the mixed gradients. The data of this experiment show that in general a very low degree oflabeling with heavy amino acids can be achieved with this organism. This is probably due to a rather high rate of basal amino acid synthesis during the labeling time. A similar situation has been reported for the slime mold Physarumpolycephalum (7, 21), for the filamentous Fomes annosus (11), and for cultured chick myotubes (4). In all of these cases, labeling with 2Hor 14C-amino-acids shows, even after very prolonged labeling times, no equilibration between the external supplied amino acids with the pool of it. Nevertheless, the degree of labeling reached in our experiments was enough to produce a significant shift in density being detectable not only by a band widening but even by double peaks in mixed gradients. Although the specific activities of nitrate reductase and the other enzymes are very different (Table I), their labeling as shown in Figures 1 and 2 was identical. From this it is obvious that the 28.5-fold increase in activity of nitrate reductase was not due to synthesis but rather to light-mediated activation of an inactive protein already present in the dark. A longer illumination up to 4 hr of the algae pregrown with deuterated amino acids results into a double peak of nitrate reductase in the gradient (Fig. 3), with one band in the position of the labeled and the other in that of the light enzyme. The same result was obtained for the other two enzymes (data not shown). This also points to the interpretation that light-mediated activation rather than de novo synthesis is due to the observed rapid increase of enzyme activity. The doubling time of nitrate reductase can be Table 1. Changes of the specific activJty of t:,ree enzysmes in Chlorella strain 211 8k after diiferent growth condition dark 1 hr ligiot increase 1 hr light+ inbioition % actiaion ) Nitrate reductase 0.5 26.8 + 2850 0.54 90 NAD-glutaste0.3 0.32. 16 0.z4 30 dehydrogenase Acid phospnatase 0.5 0.40 2 0.40 18 rel activity nit r ate red uctase (I h light) rel. activity rel activity p25 A 1p25 100] 10 30 50 fraction number 10 30 50 FIG. 1. Equilibrium density gradient sedimentation of nitrate reductase in metrizamide-D20 density gradients. Line indicates slope of gradients. Conditions: a, normal cultivation; b, precultivation in presence of heavy amino acids; c, hybrid band a + b. Induction phase: I hr of light. acid phosphatase rel activity NAD-glutanmate DH rel activity ---2 1 200 0 Ld--ri 70 30 fraction number 1250