556th MEETING, LONDON
531
The Regulation of Nitrate Reductase in the Fungus Aspergillus nidulans N. S. DUNN-COLEMAN and J. A. PATEMAN Department of Genetics, University of Glasgow, Glasgow G11 5JS, U.K.
Nitrate reductase induction by nitrate and repression by NH,+ has been studied extensively in the simple eukaryote Aspergillus nidulans (Pateman & Cove, 1967; Cove & Pateman, 1968). Wild-type cells grown on minimal medium, with 2 0 m ~ - N a N Oas~the nitrogen source,rapidly lose nitrate reductase activity when transferred either to minimal medium without N (-N medium) or to minimal medium without C and N (-CN medium) or to -CN medium plus 100m-L-glutamate as the sole N and C source. Hynes (1973), whose work we have confirmed, found that transferring pre-grown cells from 10mM-NaN03to a medium lacking a C source(-C medium) resulted in the greatest decrease in activity,even in the presence of 10m-NaN03.Cyclohexamide,an inhibitor of protein synthesis at a concentration of lOpg/ml, did not prevent the loss of activity. When cell-free extracts of wild-type cells, grown on -N medium+20m~-NaNO~, were centrifuged at 6OOOOg for 20min and incubated for periods of up to 1h with 2.65m1 of orthophosphate buffer, pH7.75,+3.5pg of FAD, there was a rapid loss of nitrate reductase activity (see Fig. 1). There was a similar loss of enzyme activity when either 1lOm-NaNO3or 3 5 m - N H was,+ added to the incubation mixture. When NADH-linked nitrate reductase extracts from Nitrobacter agilis, pre-grown on nitrate, are incubated in the absence of nitrate, rapid inactivation of the enzyme results. Herrera & Nicholas (1974) found that this inactivation was the result of over-reduction with NADH. Severalworkers also found that nitrate reductase is inactivatedby reduced
I 0
I
I
I
I
15
30
45
60
Time (min) Fig. 1. Results of various treatments on the inactivation of nitrate reductase The enzyme preparations from cells grown on nitrate were incubated in 2.65ml of orthophosphate buffer, pH7.75, with 3.5pg of FAD at 25OC. At the incubation times indicated, nitrate induction activity was determined by the method of Cove (1965). m, NADP; 0,NADPH+NH,+, A, control; A, NO3-; 0 , N H,'.
VOl. 3
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BIOCHEMICAL SOCIETY TRANSACTIONS
nicotinamide nucleotides in algae (Monreno et al., 1972; Solomonson et al., 1973). It seemed likely that the inactivation of nitrate reductase in A . nidulans might be the result of the accumulation of a particular nucleotide. Therefore nitrate reductase extracts were incubated for a period of 1h in the presence of several different nucleotides, i.e. NADPH, NADP+, NADH and NAD+. After lh incubation, 100, 47, 11 and 47 % of the initial activity remained when extracts were incubated with NADPH, NADP+, NADH and NAD+ respectively. There was a rapid loss of nitrate reductase activity after incubation with NADP+, NADH or NAD+. However, incubation with NADP+, NADH or NAD+ did not significantly alter the rate of loss of enzyme activity compared with incubation in the absence of such nucleotides. Incubation of the enzyme extract with NADPH prevented any loss of activity. We have been able to incubate nitrate reductase extracts for a period of 105 min without any significant loss of activity. The situation in Aspergillus nidulans is the reverse of that found in algae and Nitrobacter agilis; the Aspergillus enzyme is not inactivated in the presence of a nicotinamide nucleotide, but retains its activity only in the presence of NADPH. Herrera & Nicholas (1974) also found that the addition of ferrocyanide to an incubation mixture containing nitrate reductase extracts plus NADPH, resulted in the re-activation of the enzyme. In Aspergillus nidulans, addition of ferrocyanide to an incubation mixture containing nitrate reductase extract and NADPH resulted in a significant decrease in activity after an incubation period of 1h (results not shown). To find out if nitrate reductase in A. nidulans could be re-activated, NADPH was added to an incubation mixture containing only buffer, FAD and extract after an incubation period of 45min, and the activity of the enzyme was sampled at 15min timeintervals, until 2h. The results are shown in Fig. 2. Re-activation of nitrate reductase occurred rapidly after the addition of NADPH to the incubation mixture. After 105min, the initial activity of the enzyme had been reached. Therefore 100% re-activation of the enzyme is possible. The addition of NADPH after only 30min incubation resulted in a quicker re-activation of the enzyme (results not shown). Hankinson (1973) found in A . nidulans that the presence of nitrate in the growth medium affects several enzymes of the pentose phosphate pathway. Growth on nitrate results in a twofold increase in activity of glucosephosphate isomerase, an enzymewhichiscommon to thepentosephosphatepathwayand theEmbden-MayerhofParnas pathway. Hankinson (1973) also found that mutants PPPA and PPPB are characterized by poor growth on nitrate and nitrite, and have poor growth on C sources which are catabolized solely by the pentose phosphate pathway. It appears therefore that the pentose phosphate pathway in A. nidulans is an important source of theNADPH which is required for the reduction of nitrate. The results of Hynes (1973), Hankinson (1973), andour own findings, suggest that in some way NADPH activates or prevents the inactivation of nitrate reductase in A . nidulans. Consequently the concentration of NADPH is one of the factors which determine the amount of enzyme activity of those nitrate reductase molecules present in the cell. This would account for theloss of nitratereductase activity in cells transferred to medium without a C source, since the NADPH/NADP ratio would fall. It also explains the loss of nitrate reductase activity in cell-free extracts during incubation, as the initial concentrationofNADPH is decreased by nitratereductaseandotherenzyme activities. The initial concentration of NADPH in the cell-free extracts would depend onthe typeofNandCsourceonwhich thecells hadgrown. The fact thatnitratereductase activity can be restored in uitro by NADPH suggests that in A . nidulans the enzyme can be ‘turned off’ (inactivated) when the ratio of NADPH/NADP+ is low and re-activated when the ratio is high. The basic hypothesis that the NADPH/NADP+ ratio determines the amount of catalytic activity of the nitrate reductase molecule does not readily account for one of our experimental observations. If cells grown on nitrate and glucose are transferred to medium containing glucose without any N, nitrate reductase activity falls rapidly. This decrease in nitrate reductase activity is more rapid than would be expected to result just 1975
556th
z
MEETING, LONDON
0
15
30
45
533
63
75
95
105
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Time (rnin)
Fig. 2. Protective effect of NADPH on inactivation of nitrate reductase, and the reactivation of nitrate reductase with NADPH 0, 2.65ml of orthophosphate buffer (pH7.75)+3.5pg of FAD, NADPH added after 45min. M, 2.65ml of orthophosphate buffer (pH7.75)+3.5pg of FAD, NADPH added at zero time.
from the absence of the inducer nitrate. To explain this result, it is necessary to assume either than the absence of nitrate directly results in a lowered yield of NADPH from the pentose phosphate pathway, or nitrate directly or indirectly is also involved in the stabilization of nitrate reductase activity. We thank the Science Research Council for a Research Studentship to N. S. D.-C. during the period of this work. Cove, D. J. (1965) Biochim. Biophys. Acta 113,51-56 Cove, D. J. & Pateman, J. A. (1968) J. Bacferiol. 97, 1374-1378 Hankinson, 0. (1973)J. Bucreriol. 117, 1121-1130 Herrera, J. & Nicholas, D. J. D. (1974) Biochim. Biophys. Acfa368,54-60 Hynes, M. J. (1973)J. Gem Microbiof. 79,155-157 Monreno, C. G., Aparicio, P. J., PaIaciBn, E. & Losada, M. (1972) FEBS Letr. 26,ll-14 Pateman, J. A. &Cove, D. J. (1967) Nature (London) 215,1234-1237 Solomonson, L. P., Jetschmann,K. & Vennesland, B. (1973) Biochim. Biophys. Acta 309,3243
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531
The Regulation of Nitrate Reductase in the Fungus Aspergillus nidulans N. S. DUNN-COLEMAN and J. A. PATEMAN Department of Genetics, University of Glasgow, Glasgow G11 5JS, U.K.
Nitrate reductase induction by nitrate and repression by NH,+ has been studied extensively in the simple eukaryote Aspergillus nidulans (Pateman & Cove, 1967; Cove & Pateman, 1968). Wild-type cells grown on minimal medium, with 2 0 m ~ - N a N Oas~the nitrogen source,rapidly lose nitrate reductase activity when transferred either to minimal medium without N (-N medium) or to minimal medium without C and N (-CN medium) or to -CN medium plus 100m-L-glutamate as the sole N and C source. Hynes (1973), whose work we have confirmed, found that transferring pre-grown cells from 10mM-NaN03to a medium lacking a C source(-C medium) resulted in the greatest decrease in activity,even in the presence of 10m-NaN03.Cyclohexamide,an inhibitor of protein synthesis at a concentration of lOpg/ml, did not prevent the loss of activity. When cell-free extracts of wild-type cells, grown on -N medium+20m~-NaNO~, were centrifuged at 6OOOOg for 20min and incubated for periods of up to 1h with 2.65m1 of orthophosphate buffer, pH7.75,+3.5pg of FAD, there was a rapid loss of nitrate reductase activity (see Fig. 1). There was a similar loss of enzyme activity when either 1lOm-NaNO3or 3 5 m - N H was,+ added to the incubation mixture. When NADH-linked nitrate reductase extracts from Nitrobacter agilis, pre-grown on nitrate, are incubated in the absence of nitrate, rapid inactivation of the enzyme results. Herrera & Nicholas (1974) found that this inactivation was the result of over-reduction with NADH. Severalworkers also found that nitrate reductase is inactivatedby reduced
I 0
I
I
I
I
15
30
45
60
Time (min) Fig. 1. Results of various treatments on the inactivation of nitrate reductase The enzyme preparations from cells grown on nitrate were incubated in 2.65ml of orthophosphate buffer, pH7.75, with 3.5pg of FAD at 25OC. At the incubation times indicated, nitrate induction activity was determined by the method of Cove (1965). m, NADP; 0,NADPH+NH,+, A, control; A, NO3-; 0 , N H,'.
VOl. 3
532
BIOCHEMICAL SOCIETY TRANSACTIONS
nicotinamide nucleotides in algae (Monreno et al., 1972; Solomonson et al., 1973). It seemed likely that the inactivation of nitrate reductase in A . nidulans might be the result of the accumulation of a particular nucleotide. Therefore nitrate reductase extracts were incubated for a period of 1h in the presence of several different nucleotides, i.e. NADPH, NADP+, NADH and NAD+. After lh incubation, 100, 47, 11 and 47 % of the initial activity remained when extracts were incubated with NADPH, NADP+, NADH and NAD+ respectively. There was a rapid loss of nitrate reductase activity after incubation with NADP+, NADH or NAD+. However, incubation with NADP+, NADH or NAD+ did not significantly alter the rate of loss of enzyme activity compared with incubation in the absence of such nucleotides. Incubation of the enzyme extract with NADPH prevented any loss of activity. We have been able to incubate nitrate reductase extracts for a period of 105 min without any significant loss of activity. The situation in Aspergillus nidulans is the reverse of that found in algae and Nitrobacter agilis; the Aspergillus enzyme is not inactivated in the presence of a nicotinamide nucleotide, but retains its activity only in the presence of NADPH. Herrera & Nicholas (1974) also found that the addition of ferrocyanide to an incubation mixture containing nitrate reductase extracts plus NADPH, resulted in the re-activation of the enzyme. In Aspergillus nidulans, addition of ferrocyanide to an incubation mixture containing nitrate reductase extract and NADPH resulted in a significant decrease in activity after an incubation period of 1h (results not shown). To find out if nitrate reductase in A. nidulans could be re-activated, NADPH was added to an incubation mixture containing only buffer, FAD and extract after an incubation period of 45min, and the activity of the enzyme was sampled at 15min timeintervals, until 2h. The results are shown in Fig. 2. Re-activation of nitrate reductase occurred rapidly after the addition of NADPH to the incubation mixture. After 105min, the initial activity of the enzyme had been reached. Therefore 100% re-activation of the enzyme is possible. The addition of NADPH after only 30min incubation resulted in a quicker re-activation of the enzyme (results not shown). Hankinson (1973) found in A . nidulans that the presence of nitrate in the growth medium affects several enzymes of the pentose phosphate pathway. Growth on nitrate results in a twofold increase in activity of glucosephosphate isomerase, an enzymewhichiscommon to thepentosephosphatepathwayand theEmbden-MayerhofParnas pathway. Hankinson (1973) also found that mutants PPPA and PPPB are characterized by poor growth on nitrate and nitrite, and have poor growth on C sources which are catabolized solely by the pentose phosphate pathway. It appears therefore that the pentose phosphate pathway in A. nidulans is an important source of theNADPH which is required for the reduction of nitrate. The results of Hynes (1973), Hankinson (1973), andour own findings, suggest that in some way NADPH activates or prevents the inactivation of nitrate reductase in A . nidulans. Consequently the concentration of NADPH is one of the factors which determine the amount of enzyme activity of those nitrate reductase molecules present in the cell. This would account for theloss of nitratereductase activity in cells transferred to medium without a C source, since the NADPH/NADP ratio would fall. It also explains the loss of nitrate reductase activity in cell-free extracts during incubation, as the initial concentrationofNADPH is decreased by nitratereductaseandotherenzyme activities. The initial concentration of NADPH in the cell-free extracts would depend onthe typeofNandCsourceonwhich thecells hadgrown. The fact thatnitratereductase activity can be restored in uitro by NADPH suggests that in A . nidulans the enzyme can be ‘turned off’ (inactivated) when the ratio of NADPH/NADP+ is low and re-activated when the ratio is high. The basic hypothesis that the NADPH/NADP+ ratio determines the amount of catalytic activity of the nitrate reductase molecule does not readily account for one of our experimental observations. If cells grown on nitrate and glucose are transferred to medium containing glucose without any N, nitrate reductase activity falls rapidly. This decrease in nitrate reductase activity is more rapid than would be expected to result just 1975
556th
z
MEETING, LONDON
0
15
30
45
533
63
75
95
105
I20
Time (rnin)
Fig. 2. Protective effect of NADPH on inactivation of nitrate reductase, and the reactivation of nitrate reductase with NADPH 0, 2.65ml of orthophosphate buffer (pH7.75)+3.5pg of FAD, NADPH added after 45min. M, 2.65ml of orthophosphate buffer (pH7.75)+3.5pg of FAD, NADPH added at zero time.
from the absence of the inducer nitrate. To explain this result, it is necessary to assume either than the absence of nitrate directly results in a lowered yield of NADPH from the pentose phosphate pathway, or nitrate directly or indirectly is also involved in the stabilization of nitrate reductase activity. We thank the Science Research Council for a Research Studentship to N. S. D.-C. during the period of this work. Cove, D. J. (1965) Biochim. Biophys. Acta 113,51-56 Cove, D. J. & Pateman, J. A. (1968) J. Bacferiol. 97, 1374-1378 Hankinson, 0. (1973)J. Bucreriol. 117, 1121-1130 Herrera, J. & Nicholas, D. J. D. (1974) Biochim. Biophys. Acfa368,54-60 Hynes, M. J. (1973)J. Gem Microbiof. 79,155-157 Monreno, C. G., Aparicio, P. J., PaIaciBn, E. & Losada, M. (1972) FEBS Letr. 26,ll-14 Pateman, J. A. &Cove, D. J. (1967) Nature (London) 215,1234-1237 Solomonson, L. P., Jetschmann,K. & Vennesland, B. (1973) Biochim. Biophys. Acta 309,3243
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