reductase from Chlorella vulgar& by reacting with the enzyme in a manner rather similar to that of HCN. Thus vanadate, like HCN, forms an inactive complex with the reduced enzyme, and this inactivated enzyme can be reactivated rapidly by adding ferricyanide. The inactive vanadate enzyme complex is less stable than the inactive HCN complex, and the two can be distinguished by the fact that EDTA causes a partial reactivation of the former, but not of the latter. Vanadate can also cause an increase in HCN formation by intact Chlorella vulgaris cells. When these cells were incubated with vanadate, their nitrate reductase was reversibly inactivated, and all of this inactive enzyme could be shown to be the HCN complex rather than the vanadate complex. When HCN and vanadate are both present, the HCN-inactivated enzyme, being more stable, will be formed in preference to the vanadate-inactivated enzyme. Key words: Chlorella - Cyanide - Nitrate reductase - Vanadium.
Tungsten and vanadium have been shown to interfere in vivo with the action of the molybdoprotein nitrate reductase. This phenomenon has been demonstrated and studied in plants (Wray and Filner, 1970; Notton and Hewitt, 1971 a; Notton and Hewitt, 1971b; Buczek, 1973; Norton et al., 1974), algae (Vega etal., 1971), and in the mold Neurospora crassa (Subramanian and Sorger, 1972; Lee et al., 1974; Schloemer Abbrev&tion: EDTA=ethylenediamine tetraacetate
and Garrett, 1974). With spinach it has been shown that tungsten is incorporated in vivo into nitrate reductase, presumably to give a tungsten analogue of the molybdoprotein (Notton and Hewitt, 1971b). This tungsten analogue has an undiminished diaphorase activity (reduction of cytochrome c by NADH), but cannot activate nitrate. Tungsten, however, has no effect on nitrate reductase in vitro. In a comparative study of the effects of tungsten and vanadium on nitrate reductase of N. c r a s s a ( L e e et al., 1974), it was reported that vanadium salts also had no effect on partially purified nitrate reductase in vitro, and because of the similar inhibitory effect of both metal ions in vivo, the conclusion was drawn that vanadium probably acts like tungsten in the sense that it forms a vanadium enzyme analogue which cannot activate nitrate. In contrast, Buczek (1973) reported that vanadium salts inhibited the nitrate reductase of tomato seedlings both in vitro and in vivo. The inhibited enzyme could be reactivated by treatment with EDTA or by passage through a Sephadex column. Such reversal of inhibition is incompatible with the formation of a metalloprotein analogue. Thus, the results reported with N. crassa (Lee et al., 1974) and with tomato seedlings (Buczek, 1973) seem quite contradictory. The present studies were prompted by this contradiction. We will show here that vanadium inhibits the nitrate reductase of the alga Chlorella vulgaris both in vivo and in vitro, and that this inhibition can be reversed without any addition of molybdenum. The action of vanadium will be shown to resemble the action of HCN rather than tungsten.
Materials and Methods NaVO3. HzO; NazMoO4-2H20 and NazWO4.2H20 used were purchased from Merck. NADH (grade I) was obtained from Boehringer.
C.S. Ramadoss: Effect of Vanadium on Nitrate Reductase
Growth of Algae
Cultures of C. vulgaris were maintained with nitrate as the sole source of nitrogen, as previously described (Vennesland and Jetschmann, 1971). The culture medium contained 0.02 M MgSO4, 0.018M KHzPO4, 0.034M NaC1, 0.020M KNO3, 0.002M Ca(NOa)2 and the following microelements: 14 ~tM FeSO4, 20 gM Fe(NO3)a, 341-tM H3BO3, 0.61 gM ZnSO4, 9.4gM MnSO4, 0.26gM CuSO4, 0.013gM (NH4)6MoTO2,, 0.07lsM COSO4, 0.08 gM NiSO4, 0.06 gM Na2WO4, 0.04 gM KCr(SO4)2, 0.08 gM VOSO4, 1 gM NaVO3. After growth at 20-22~ for 24h at a light intensity of 30,000 lx in 5% CO2 in air, the cell density increased from 0.4 to 3-4 gl cells per ml.
Cyanide associated with nitrate reductase in the protamine sulfate fraction was determined by activation of the enzyme by nitrate and estimation of the amount of cyanide released, as described previously (Lorimer et al., 1974).
Experimental Procedure For the experiments in Table 1, the cells were harvested and washed twice with medium A, which contained 0.02 M MgSO4, 0.018 M KHzPO4, 0.01 M Na2HPO4, 0.002 M CaClz, 0.034 M NaC1. The pH of the medium was 6.3. The washed cells were resuspended to give 3 4 gl cells per ml in medium A (pH 6.3) containing in addition 10 mM KNO3 and the following microelements: 14 gM FeSO4, 20 gM Fe(NO3)3, 34 gM H3BO3, 0.61 gM ZnSO4, 9.4 ~tM MnSO4, 0.26 gM CuSO4. This cell suspension was divided into two parts: to one, vanadate (NaVQ) or tungstate (Na2WO4) was added to give a final concentration of 2 mM; the other part served as the control. The flasks (500 ml) containing these cell suspensions were returned to standard growth conditions (light 30,000 lx, 5% CO2 in air). After 24 h of growth, the cell density in the control, vanadate and tungstate treated flasks were in the range of 6-8 gl cells per ml. The cells were harvested, washed once with 0.01 M phosphate buffer, pH 6.9 and resuspended in the same buffer to give a cell density of 250 ~1 cells per ml.
French Press Extract The cell suspension was disrupted with a French Pressure cell press (Aminco) and precooled in ice, at 10,000 p.s.i. The French press extract was centrifuged at 16,000 g for 10 min. The deep green supernatant was decanted and tested immediately. Part of the extract was kept frozen at - 2 0 ~ C until further use.
Protamine Sulfate Fractionation Cell extract, kept frozen at - 2 0 ~ C for about a week, was thawed and centrifuged at 16,000 g for 10 min. The clear pale supernatant was used for protamine sulfate fractionation. To 75 ml of the extract, 0.1 vol of 2% protamine sulfate (Merck) was added and mixed well. After 5 min standing in ice, the precipitated material was removed by centrifugation at 12,000g for 10 rain. To the supernatant another 0.2 vol of protamine sulfate (2%) was added, and the mixture was stirred for 5 min. The precipitated proteins were collected by centrifugation at 16,000 g for 10 rain. Alternatively, the supernatant solution was brought to 0.5 saturation with finely ground ammonium sulfate. The solution was stirred for 15 min, and then stored in an ice bath for 2 h. The precipitated proteins were collected by centrifugation as before. The protein was resuspended in 0.1 M potassium phosphate buffer, pH 6.9. The insolubles were removed by centrifugation at 30,000g for 15 min. The clear brown-yellow supernatant was used for further analysis.
Purification of Nitrate Reduetase Nitrate reductase from C. vulgaris was purified essentially according to the procedure of Solomonson (1975). The enzyme used in these studies had specific activity in the range of 85 90 units per mg protein. Purified enzyme was usually stored at - 2 0 ~ in 0.08 M potassium phosphate buffer, pH 6.9, containing 40% (v/v) glycerol, 0.14ram mercaptoethanol, 0.1 mM EDTA and 0.03 mM chloramphenicol. A sample of purified nitrate reductase from Chlorella fusca was prepared as described by Gewitz et al. (1978). One unit of enzyme activity is that amount with catalyzes the formation of 1 gmol of nitrite per min at 20~ C under conditions previously defined (Solomonson and Vennesland, 1972; Solomonson, 1974). NADH: cytochrome c reductase was also assayed at 20~ C as previously described (Solomonson and Vennesland, 1972; Solomonson, 1974).
Results and Discussion
Effect of Vanadate on PuriJi'ed Enzyme Purified nitrate reductase from C. vulgaris was effectively inhibited by added vanadate (NaVO3). The full inhibitory effect was not immediate but developed with time as the result of an interaction of vanadate with the reduced enzyme. The latter is formed by addition of NADH without nitrate. Figure 1 shows the development of this vanadate inhibition with time at different vanadate concentrations in the presence of NADH. Almost complete inhibition was developed within 5 rain at room temperature by 50 gM NaVO3. If NADH was omitted, vanadate at this concentration had no inhibitory effect. Similarly, NADH alone had no inhibitory effect in the absence of vanadate. The purified nitrate reductase of C.fusca responded to vanadate very much like the C. vulgaris enzyme. It has been claimed that this enzyme is inactivated by NADH alone and that the inactivation is potentiated by ADP (Maldonado et al., 1973), but these results have not been substantiated with purified enzyme. It may be of interest in this connection, however, that ATP preparations derived from muscle have been reported to be contaminated with vanadium (Cantley et al., 1977). When vanadate was added directly to the enzyme assay mixture with NADH and N O 3 , inhibition of the enzyme also developed with time, but higher vanadate concentrations were required to effect a given level of inhibition in a given time than when the vanadate was incubated with reduced enzyme, as
C.S. Ramadoss : Effect of Vanadium on Nitrate Reductase
was constant. In the presence of vanadat% the curves were concave up, showing that the rate was declining. Even with 0.3 mM vanadate, a detectable oxidation of NADH by NO~- occurred before full inhibition of the enzyme was established.
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