Planta (Berl.) 122, 293--297 (1975) 9 by Springer-Verlag 1975
Light-Regulation of Enzyme Activity in Anacystis nidulans (Richt.) Jeffrey X. Duggan and Louise E. Anderson Department of Biological Sciences, University of Illinois at Chicago Circle, Box 4348, Chicago, Illinois 60690, USA Received 31 October; accepted 21 November, 1974 ~ummary. The effect of light on the levels of activity of six enzymes which are lightmodulated in higher plants was examined in the photosynthetic proearyot Anacysti8 nidulans. Ribulose-5-phosphate kinase (EC2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inaetivated. The enzymes fructose-l,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-l,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1,2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyeeraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathways in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.
I n higher plants the synthesis and degradation of glucose is controlled by light. Four enzymes of the reductive pentose phosphate cycle, and in Ca-plants two additional enzymes of the C4 dicarboxylic acid CO s fixation pathway, are activated in the intact leaf by a light-dependent process, and two enzymes of glucose degradation are inactivated. I n most cases dithiothreitol treatment affects the activity of these enzymes in extracts as well, and it is thought that light-modulation may involve reduction of disulfide bonds of the regulated enzymes (see Anderson, 1974a). Most studies of light-activation have involved angiosperms, although lightactivation of I~ADP-linked glyceraldehyde-3-P dehydrogenase has been reported to occur in several species of algae (Ziegler and Ziegler, 1967; Ziegler et al., 1967). The purpose of the present experiments was to determine whether light-modulation of the activity of enzymes of carbon metabolism occurs in procaryots as well as in eucaryots. We now report that the reductive pentose phosphate pathway enzyme ribulose-5-P kinase is light-activated in the blue green alga Anacystis nidulans and t h a t the oxidative pentose phosphate pathway enzyme glucose-6-P dehydrogenase is light-inactivated. We were not able to detect activation of reductive pentose phosphate pathway enzymes in the photosynthetic bacterium Rhodospirillum rubrum. 20
P l a n t a (BerL), Vol. 122
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J . X . Duggan and L. E. Anderson
Materials and Methods Growth Conditions. Anacystis nidulans (Richt.), obtained from Prof. G. Benjamin Bouck, was cultured in medium "C" of Kratz and Myers (1955) with the pH adjusted to 8.0 prior to sterilization. A 10 % inoculum was used. Cultures were maintained at 35~ C in cotton stoppered flasks, 1/3 filled, under four 30 W, 150 volt General Electric reflector bulbs (1.15 • 105 ergs cm-2 s-1) 30 cm distant. The flasks were shaken at 125 rpm on a gyrorotary shaker and were continuallyflushed with CO2-enriched air produced by bubbling air through saturated Na2COa solution. After 60 to 75 h of growth log phase cultures (A600:~ were harvested by centrifugation, washed once with 50 mM potassium phosphate buffer pH 7.0, then with 100 mM Tris HC1 pH 7.6, resuspended in a minimal volume of the Tris buffer and returned to the shaker. Light-treated cultures were exposed to light. ])ark-treated cultures were wrapped in foil and shaken in the dark. After 45 min extracts were prepared by passing the cells through a French pressure cell at 20000psi and after centrifugation (20 min 40000 g) activity of enzymes in the supernatant solution was determined. Dithiothreitol treatment consisted of making extracts from dark-treated cells 50 m ~ in dithiothreitol and allowing the extracts to stand on ice for at least 30 min prior to activity determinations.
Enzyme Assays. NAD- and NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12, EC 1.1.1.13) were assayed by the method of Wu and Racker (1959; see Anderson et al., 1974); ribulose-5-P kinase (EC 2.7.1.19) by the method of Hurwitz et al. (1956) with ATP final concentration 0.1 mM (see Anderson, 1973); NADP-linked malic dehydrogenase (EC 1.1.1.82) as described by Hatch and Slack (1969; see Anderson et al., 1974), sedoheptulose-l,7-di P phosphatase as described by Anderson (1974b) except that the reaction mixture was 2 mM in EI)TA; fructose-l,6-diP phosphate (EC 3.1.3.11) by the method of Smillie (1964) without EDTA; glucose-6-P dehydrogenase (EC 1.1.1.49) as described by Mute and Uritani (1970; see Anderson et al., 1974) ; and P-fructokinase (EC 2.7.1.11) by the method of Ling et al. (1966; see Kachru and Anderson, 1975). Change in Aa~0 was followed at 25~ C using a Gilford 2 400 recording speetrophotometer, except in the case of the phosphatases, where phosphate released was measured after 30 rain. Controls were run in all cases without the carbon substrates and, in the case of the kinases, an additional control was run without ATP. Protein Determination. Protein was precipitated from the extracts with acetone and estimated by the biuret method (see Anderson and Advani, 1970). Since some As~0 material in the A. nidulans extracts could not be removed by acetone extraction, duplicate acetone precipitates were dissolved in 3.3% NaOH, the A550 of this solution was measured, and A55o in biuret solution corrected for this absorption. As~0 in base accounted for at most 20% of the As~o reading in biuret solution. Results and Discussion L i g h t - t r e a t m e n t activates ribulose-5-P kinase a n d i n a c t i v a t e s glueose-6-P dehydrogenase i n Anacystis nidu[ans (see T a b l e 1). I n crude extracts b o t h of these e n z y m e s are affected b y t r e a t m e n t with dithiothreitol. S t e a d y - s t a t e exp e r i m e n t s with whole cells of Aphanocapsa indicate t h a t these two enzymes are light-regulated i n this blue-green alga as well (Pelroy a n d Bassham, 1972). R i b u l o s e - l , 5 - d i P i n h i b i t s glncose-6-P dehydrogenase from Anacystis (Pelroy et al., 1972) a n d 6-P-gluconate i n h i b i t s ribulose-diphosphate carboxylase ( T a b i t a a n d M c F a d d e n , 1972). The effect of l i g h t - a c t i v a t i o n of t h e kinase o n t h e operation of t h e reductive pentose p h o s p h a t e p a t h w a y will be reinforced b y l i g h t - i n a c t i v a t i o n of t h e dehydrogenase a n d t h e r e s u l t a n t drop i n t h e levels of t h e carboxylase inhibitor. Ribulose-5-P kinase from pea shoots is i n h i b i t e d b y 6-P-gluconate (Anderson, 1973). I f t h e kinase from Anacystis is also affected b y 6-P-glueonate t h e n t h e kinase a n d the dehydrogenase will be a c t i v a t e d a n d i n h i b i t e d reciprocally. Clearly, as is t h e case i n higher p l a n t s (see K a c h r u a n d Anderson, 1975) extensive
Light-Regulation of Enzyme Activity in Anacystis nldulans (Richt.)
295
Table 1. Effect of light and dithiothreitol treatment on the activity of A. nidulans reduetive and oxidative pentose phosphate cycle enzymes Light-grown cultures were harvested and resuspended in buffer either in foil wrapped flasks (dark-treated) or in flasks exposed to light. After 45 rain the cell suspensions were disrupted. Dithiothreitol-treatment consisted of adding the reducing agent to a concentration of 50 mM to the extract of the dark-treated cells. Specific activities (nmol min-x mg protein-x) are based on protein in the 40000 g supernatant solution. Enzyme
Treatment dark
Stimulation x-fold light
dithiothreitol
light
dithiothreitol
Fructose-l,6-diP phosphatase
47.0 32.1
48.9 33.0
46.1 39.6
---
-1.2
Sedoheptulose 1.7-dip phosphatase a
9.4 30.4
7.3 24.4
63.8 219
---
6.8 7.2
40.9 97.2 106
43.1 315 162
NAD-linked glyceraldeyhde-3-P dehydrogenase
2.9 1.4
1.2 i.9
NADP-linked glyceraldehyde-3-P dehydrogenase
11.1 5.7
8.8 7.0
Ribulose-5-P kinase
126 576 333 10.5 3.4 213 129
1.1 3.2 1.5 ---1.2
3.1 5.9 3.1 3.6 2.4 19 23
% Inhibition
Glucose-6-P dehydrogenase
7.25 4.5
5.4 3.8
1.3 0.64
light
dithiothreitol
26 16
82 86
a No activity was detected with sedoheptulose-7-P as substrate.
synthesis a n d d e g r a d a t i o n of glucose-6-P will n o t occur s i m u l t a n e o u s l y i n this blue green alga. N e i t h e r of the phosphatases tested appeared to be light a c t i v a t e d i n Anacystis. Since a c t i v a t i o n of fructose-I, 6-diP phosphatase is seen only w h e n Mg ~+ levels i n t h e assay m i x t u r e are low it is possible t h a t a c t i v a t i o n would n o t have been detected i n these experiments. Sedoheptulose-l,7-diP phosphatase a c t i v i t y i n crude extracts was e n h a n c e d b y dithiothreitol t r e a t m e n t . A c t i v a t i o n of sedoh e p t u l o s e - l , 7 - d i P phosphatase i n pea leaves b y light a n d dithiothreitol has been reported (Anderson, 1974 b). B o t h HAD- a n d N A D P - l i n k e d glyceraldehyde-3-P dehydrogenase were act i v a t e d b y dithiothreitol b u t n e i t h e r was light activated. I t m a y be t h a t b o t h t h e dehydrogenase(s) a n d the phosphatases are controlled b y t h e n a t u r e of t h e available c a r b o n sources r a t h e r t h a n b y light i n this blue green alga, as is glycera l d e h y d e - 3 - P dehydrogenase i n Chromatium (Hudock a n d Fuller, 1965). No P-fructokinase or N A D P - l i n k e d malie dehydrogenase a c t i v i t y was detected i n Anacystis extracts. 20*
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I n a similar series of experiments with the photosynthetic bacterium Rhodo. spiriUum rubrum, strain S-1, grown on the butyrate media of Tabita and McFadden (1974), we were unable to detect light activation of a n y of the enzymes tested above although dithiothreitol-treatment did stimulate the activity of NAD-linked glyceraldehyde-3-P dehydrogenase 12-fold (data not shown). Dithiothreitolt r e a t m e n t did not activate ribulose-5-P kinase or fructose-l,6-diP phosphatase. No sedoheptulose-l,7-diP phosphatase, P-fruetokinase, glueose-6-P dehydrogenase, NADP-linked malic dehydrogenase or NADP-Hnked glyceraldehyde dehydrogenase activity was detected. Activity levels for NAD-linked glyeeraldehyde-3-P dehydrogenase, fruetose-l,6-diP phosphatase and ribulose-5-P kinase were intermediate between levels observed for H~, COs-grown cells and acetate-grown cells previously (Anderson and Fuller, 1967). This organism is sensitive to 03 under photosynthetic conditions. I n these experiments the cultures were therefore lightor dark-treated and then harvested and disrupted. I t is possible t h a t inactivation of the activated enzymes, as a result of darkness or of 0 3, occurred during harvesting of the cells. Light apparently controls the operation of the oxidative and reduetive pentose phosphate pathways in blue-green alga Anacystis through inactivation of glucose6-I) dehydrogenase and activation of ribulose-5-P kinase b y a process which m a y involve reduction of the enzyme proteins. Modulation of the activities of these and several additional enzymes occurs in higher plants. We were unable to show lightactivation of any of these enzymes in the photosynthetic bacterium R. rubrum. Modulation of enzyme activity b y the available energT source will probably be most efficient in obligate autotrophs. I t m a y be t h a t light-modulation evolved concomitantly with obligate autotrophy in photosynthetic organisms. We thank Kyung-Eun Yoon Park for doing the glueose-6-P dehydrogenase assays. This work was supported by grant GB 28160-A1 from the United States National Science Foundation. References Anderson, L.E.: Regulation of pea leaf ribulose-5-phosphate kinase activity. Biochim. biophys. Acta (Amst.) 821, 484-488 (1973) Anderson, L. E. : Light modulation of the activity of carbon metabolism enzymes. In: Proceedings, I I I International Congress on Photosynthesis Research, Rehovot, in press Avron, M., ed., Amsterdam: A.S.P. Biological and Medical Press, B.V. 1974a Anderson, L. E. : Activation of pea leaf chloroplast sedoheptulose 1,7-diphosphate phosphatase by light and dithiothreitol. Biochem. biophys. Res. Commun. 59, 907-913 (1974b) Anderson, L.E., Advani, V. R.: Chloroplast and eytoplasmie enzymes. Three distinct isoenzymes associated with the reductive pentose phosphate cycle. Plant Physiol. 45,583-585 (1970) Anderson, L. E., Fuller, R. C.: Photosynthesis in RhodospirillumrubrumIII. Metabolic control of reductive pentose phosphate and tricarboxytic acid cycle enzymes. Plant Physiol. 42, 497-502 (1967) Anderson, L. E., Ng, T-C. L., Park, K. E. u : Inactivation of pea leaf chloroplastic and cytoplasmic glucose 6-phosphate dehydrogenases by light and dithiothreitol. Plant Physiol. 53, 835-839 (1974) Hatch, M.D., Slack, C. R.: NADP-Specifie malate dehydrogenase and glycerate kinase in leaves and evidence for their location in chloroplasts. Biochem. biophys. Res. Commun. 34, 589-593 (1969)
Light-Regulation of Enzyme Activity in Anacystis nidulans (Richt.)
297
Hudock, G.A., Fuller, R. C.: Control of triosephosphate dehydrogenase in photosynthesis. Plant Physiol. 40, 1205-1211 (1965) Hurwitz, J., Weissbach, A., Horecker, B. L., Smyrniotis, P. Z. : Spinach phosphoribulokinase. J. biol. Chem. 218, 769-783 (1956) Kachru, R. B., Anderson, L. E. : Inactivation of pea leaf phosphofrucfokinase by light and dithiothreitol. Plant Physiol., in press (1975) Kratz, W. A., Myers, J. : Nutrition and growth of several blue-green algae. Amer. ft. Bot. 42, 282-287 (1955) Ling, K. lt., Paetkan, V., Marcus, R., Lardy, It. A. : Phosphofructokinasc I. Skeletal muscle. In: Methods in enzymology, vol. 9, p. 425429, W. A. Wood, ed. New York: Academic Press 1966 Muto, S., Uritani, I.: Glucose 6-phosphate dehydrogenase from sweet potato. Plant Cell Physiol. 11, 767-776 (1970) Pelroy, R . A . , Bassham, J . A . : Photosynthetic and dark carbon metabolism in unicellular blue-green algae. Arch. Mikrobiol. 86, 25-38 (1972) Pelroy, R. A., Rippka, R., Stanier, R. Y. : The metabolism of glucose by unicellular blue-green algae. Arch. Mikrobiol. 87, 303-322 (1972) Smillie, R. M.: Plant fructose-l,6-diphosphatasc. In: Fructose-l,6-diphosphatase and its role in gluconeogenesis, p. 31-41, McGilvery, R. W., Pogell, B. M., eds. Baltimore: Port City Press 1964 Tabita, F . R . , McFadden, B.A.: Regulation of ribulose-l,5-diphosphate carboxylasc by 6-phospho-D-gluconate. Biochem. biophys. Res. Commun. 48, 1153-1159 (1972) Tabita, F . R . , McFadden, B.A.: D-Ribulose 1,5-diphosphate carboxylase from Rhodospirillum rubrum I Levels, purification, and effects of metallic ions. J. biol. Chem. 249, 3453-3468 (1974) Wu, R., Racker, E. : Regulatory mechanisms in carbohydrate metabolism I I I Limiting factors in glycolysis of ascites tumor cells. J. biol. Chem. 284, 1029-1035 (1959) Ziegler, H., Ziegler, I.: Die lichtindnzierte Aktivit~tssteigerung der NADP+-abh~ngigen Glyccrinaldehyd-3-phosphat-dehydrogenase. V. Das Verhalten yon Mceresalgen. Planta (Berl.) 72, 162-169 (1967) Ziegler, H., Ziegler, I., Beth, K.: Die lichtinduzierte Aktivit~tssteigerung der NADP+abh~ingigen Glyccrinaldehyd-3-Phosphat-Dehydrogenase. VI. Der Einflull des Zellkernes auf den Effekt. Planta (Berl.) 72, 247-251 (1967)
Light-Regulation of Enzyme Activity in Anacystis nidulans (Richt.) Jeffrey X. Duggan and Louise E. Anderson Department of Biological Sciences, University of Illinois at Chicago Circle, Box 4348, Chicago, Illinois 60690, USA Received 31 October; accepted 21 November, 1974 ~ummary. The effect of light on the levels of activity of six enzymes which are lightmodulated in higher plants was examined in the photosynthetic proearyot Anacysti8 nidulans. Ribulose-5-phosphate kinase (EC2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inaetivated. The enzymes fructose-l,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-l,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1,2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyeeraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathways in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.
I n higher plants the synthesis and degradation of glucose is controlled by light. Four enzymes of the reductive pentose phosphate cycle, and in Ca-plants two additional enzymes of the C4 dicarboxylic acid CO s fixation pathway, are activated in the intact leaf by a light-dependent process, and two enzymes of glucose degradation are inactivated. I n most cases dithiothreitol treatment affects the activity of these enzymes in extracts as well, and it is thought that light-modulation may involve reduction of disulfide bonds of the regulated enzymes (see Anderson, 1974a). Most studies of light-activation have involved angiosperms, although lightactivation of I~ADP-linked glyceraldehyde-3-P dehydrogenase has been reported to occur in several species of algae (Ziegler and Ziegler, 1967; Ziegler et al., 1967). The purpose of the present experiments was to determine whether light-modulation of the activity of enzymes of carbon metabolism occurs in procaryots as well as in eucaryots. We now report that the reductive pentose phosphate pathway enzyme ribulose-5-P kinase is light-activated in the blue green alga Anacystis nidulans and t h a t the oxidative pentose phosphate pathway enzyme glucose-6-P dehydrogenase is light-inactivated. We were not able to detect activation of reductive pentose phosphate pathway enzymes in the photosynthetic bacterium Rhodospirillum rubrum. 20
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J . X . Duggan and L. E. Anderson
Materials and Methods Growth Conditions. Anacystis nidulans (Richt.), obtained from Prof. G. Benjamin Bouck, was cultured in medium "C" of Kratz and Myers (1955) with the pH adjusted to 8.0 prior to sterilization. A 10 % inoculum was used. Cultures were maintained at 35~ C in cotton stoppered flasks, 1/3 filled, under four 30 W, 150 volt General Electric reflector bulbs (1.15 • 105 ergs cm-2 s-1) 30 cm distant. The flasks were shaken at 125 rpm on a gyrorotary shaker and were continuallyflushed with CO2-enriched air produced by bubbling air through saturated Na2COa solution. After 60 to 75 h of growth log phase cultures (A600:~ were harvested by centrifugation, washed once with 50 mM potassium phosphate buffer pH 7.0, then with 100 mM Tris HC1 pH 7.6, resuspended in a minimal volume of the Tris buffer and returned to the shaker. Light-treated cultures were exposed to light. ])ark-treated cultures were wrapped in foil and shaken in the dark. After 45 min extracts were prepared by passing the cells through a French pressure cell at 20000psi and after centrifugation (20 min 40000 g) activity of enzymes in the supernatant solution was determined. Dithiothreitol treatment consisted of making extracts from dark-treated cells 50 m ~ in dithiothreitol and allowing the extracts to stand on ice for at least 30 min prior to activity determinations.
Enzyme Assays. NAD- and NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12, EC 1.1.1.13) were assayed by the method of Wu and Racker (1959; see Anderson et al., 1974); ribulose-5-P kinase (EC 2.7.1.19) by the method of Hurwitz et al. (1956) with ATP final concentration 0.1 mM (see Anderson, 1973); NADP-linked malic dehydrogenase (EC 1.1.1.82) as described by Hatch and Slack (1969; see Anderson et al., 1974), sedoheptulose-l,7-di P phosphatase as described by Anderson (1974b) except that the reaction mixture was 2 mM in EI)TA; fructose-l,6-diP phosphate (EC 3.1.3.11) by the method of Smillie (1964) without EDTA; glucose-6-P dehydrogenase (EC 1.1.1.49) as described by Mute and Uritani (1970; see Anderson et al., 1974) ; and P-fructokinase (EC 2.7.1.11) by the method of Ling et al. (1966; see Kachru and Anderson, 1975). Change in Aa~0 was followed at 25~ C using a Gilford 2 400 recording speetrophotometer, except in the case of the phosphatases, where phosphate released was measured after 30 rain. Controls were run in all cases without the carbon substrates and, in the case of the kinases, an additional control was run without ATP. Protein Determination. Protein was precipitated from the extracts with acetone and estimated by the biuret method (see Anderson and Advani, 1970). Since some As~0 material in the A. nidulans extracts could not be removed by acetone extraction, duplicate acetone precipitates were dissolved in 3.3% NaOH, the A550 of this solution was measured, and A55o in biuret solution corrected for this absorption. As~0 in base accounted for at most 20% of the As~o reading in biuret solution. Results and Discussion L i g h t - t r e a t m e n t activates ribulose-5-P kinase a n d i n a c t i v a t e s glueose-6-P dehydrogenase i n Anacystis nidu[ans (see T a b l e 1). I n crude extracts b o t h of these e n z y m e s are affected b y t r e a t m e n t with dithiothreitol. S t e a d y - s t a t e exp e r i m e n t s with whole cells of Aphanocapsa indicate t h a t these two enzymes are light-regulated i n this blue-green alga as well (Pelroy a n d Bassham, 1972). R i b u l o s e - l , 5 - d i P i n h i b i t s glncose-6-P dehydrogenase from Anacystis (Pelroy et al., 1972) a n d 6-P-gluconate i n h i b i t s ribulose-diphosphate carboxylase ( T a b i t a a n d M c F a d d e n , 1972). The effect of l i g h t - a c t i v a t i o n of t h e kinase o n t h e operation of t h e reductive pentose p h o s p h a t e p a t h w a y will be reinforced b y l i g h t - i n a c t i v a t i o n of t h e dehydrogenase a n d t h e r e s u l t a n t drop i n t h e levels of t h e carboxylase inhibitor. Ribulose-5-P kinase from pea shoots is i n h i b i t e d b y 6-P-gluconate (Anderson, 1973). I f t h e kinase from Anacystis is also affected b y 6-P-glueonate t h e n t h e kinase a n d the dehydrogenase will be a c t i v a t e d a n d i n h i b i t e d reciprocally. Clearly, as is t h e case i n higher p l a n t s (see K a c h r u a n d Anderson, 1975) extensive
Light-Regulation of Enzyme Activity in Anacystis nldulans (Richt.)
295
Table 1. Effect of light and dithiothreitol treatment on the activity of A. nidulans reduetive and oxidative pentose phosphate cycle enzymes Light-grown cultures were harvested and resuspended in buffer either in foil wrapped flasks (dark-treated) or in flasks exposed to light. After 45 rain the cell suspensions were disrupted. Dithiothreitol-treatment consisted of adding the reducing agent to a concentration of 50 mM to the extract of the dark-treated cells. Specific activities (nmol min-x mg protein-x) are based on protein in the 40000 g supernatant solution. Enzyme
Treatment dark
Stimulation x-fold light
dithiothreitol
light
dithiothreitol
Fructose-l,6-diP phosphatase
47.0 32.1
48.9 33.0
46.1 39.6
---
-1.2
Sedoheptulose 1.7-dip phosphatase a
9.4 30.4
7.3 24.4
63.8 219
---
6.8 7.2
40.9 97.2 106
43.1 315 162
NAD-linked glyceraldeyhde-3-P dehydrogenase
2.9 1.4
1.2 i.9
NADP-linked glyceraldehyde-3-P dehydrogenase
11.1 5.7
8.8 7.0
Ribulose-5-P kinase
126 576 333 10.5 3.4 213 129
1.1 3.2 1.5 ---1.2
3.1 5.9 3.1 3.6 2.4 19 23
% Inhibition
Glucose-6-P dehydrogenase
7.25 4.5
5.4 3.8
1.3 0.64
light
dithiothreitol
26 16
82 86
a No activity was detected with sedoheptulose-7-P as substrate.
synthesis a n d d e g r a d a t i o n of glucose-6-P will n o t occur s i m u l t a n e o u s l y i n this blue green alga. N e i t h e r of the phosphatases tested appeared to be light a c t i v a t e d i n Anacystis. Since a c t i v a t i o n of fructose-I, 6-diP phosphatase is seen only w h e n Mg ~+ levels i n t h e assay m i x t u r e are low it is possible t h a t a c t i v a t i o n would n o t have been detected i n these experiments. Sedoheptulose-l,7-diP phosphatase a c t i v i t y i n crude extracts was e n h a n c e d b y dithiothreitol t r e a t m e n t . A c t i v a t i o n of sedoh e p t u l o s e - l , 7 - d i P phosphatase i n pea leaves b y light a n d dithiothreitol has been reported (Anderson, 1974 b). B o t h HAD- a n d N A D P - l i n k e d glyceraldehyde-3-P dehydrogenase were act i v a t e d b y dithiothreitol b u t n e i t h e r was light activated. I t m a y be t h a t b o t h t h e dehydrogenase(s) a n d the phosphatases are controlled b y t h e n a t u r e of t h e available c a r b o n sources r a t h e r t h a n b y light i n this blue green alga, as is glycera l d e h y d e - 3 - P dehydrogenase i n Chromatium (Hudock a n d Fuller, 1965). No P-fructokinase or N A D P - l i n k e d malie dehydrogenase a c t i v i t y was detected i n Anacystis extracts. 20*
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J . X . Duggan and L. E. Anderson
I n a similar series of experiments with the photosynthetic bacterium Rhodo. spiriUum rubrum, strain S-1, grown on the butyrate media of Tabita and McFadden (1974), we were unable to detect light activation of a n y of the enzymes tested above although dithiothreitol-treatment did stimulate the activity of NAD-linked glyceraldehyde-3-P dehydrogenase 12-fold (data not shown). Dithiothreitolt r e a t m e n t did not activate ribulose-5-P kinase or fructose-l,6-diP phosphatase. No sedoheptulose-l,7-diP phosphatase, P-fruetokinase, glueose-6-P dehydrogenase, NADP-linked malic dehydrogenase or NADP-Hnked glyceraldehyde dehydrogenase activity was detected. Activity levels for NAD-linked glyeeraldehyde-3-P dehydrogenase, fruetose-l,6-diP phosphatase and ribulose-5-P kinase were intermediate between levels observed for H~, COs-grown cells and acetate-grown cells previously (Anderson and Fuller, 1967). This organism is sensitive to 03 under photosynthetic conditions. I n these experiments the cultures were therefore lightor dark-treated and then harvested and disrupted. I t is possible t h a t inactivation of the activated enzymes, as a result of darkness or of 0 3, occurred during harvesting of the cells. Light apparently controls the operation of the oxidative and reduetive pentose phosphate pathways in blue-green alga Anacystis through inactivation of glucose6-I) dehydrogenase and activation of ribulose-5-P kinase b y a process which m a y involve reduction of the enzyme proteins. Modulation of the activities of these and several additional enzymes occurs in higher plants. We were unable to show lightactivation of any of these enzymes in the photosynthetic bacterium R. rubrum. Modulation of enzyme activity b y the available energT source will probably be most efficient in obligate autotrophs. I t m a y be t h a t light-modulation evolved concomitantly with obligate autotrophy in photosynthetic organisms. We thank Kyung-Eun Yoon Park for doing the glueose-6-P dehydrogenase assays. This work was supported by grant GB 28160-A1 from the United States National Science Foundation. References Anderson, L.E.: Regulation of pea leaf ribulose-5-phosphate kinase activity. Biochim. biophys. Acta (Amst.) 821, 484-488 (1973) Anderson, L. E. : Light modulation of the activity of carbon metabolism enzymes. In: Proceedings, I I I International Congress on Photosynthesis Research, Rehovot, in press Avron, M., ed., Amsterdam: A.S.P. Biological and Medical Press, B.V. 1974a Anderson, L. E. : Activation of pea leaf chloroplast sedoheptulose 1,7-diphosphate phosphatase by light and dithiothreitol. Biochem. biophys. Res. Commun. 59, 907-913 (1974b) Anderson, L.E., Advani, V. R.: Chloroplast and eytoplasmie enzymes. Three distinct isoenzymes associated with the reductive pentose phosphate cycle. Plant Physiol. 45,583-585 (1970) Anderson, L. E., Fuller, R. C.: Photosynthesis in RhodospirillumrubrumIII. Metabolic control of reductive pentose phosphate and tricarboxytic acid cycle enzymes. Plant Physiol. 42, 497-502 (1967) Anderson, L. E., Ng, T-C. L., Park, K. E. u : Inactivation of pea leaf chloroplastic and cytoplasmic glucose 6-phosphate dehydrogenases by light and dithiothreitol. Plant Physiol. 53, 835-839 (1974) Hatch, M.D., Slack, C. R.: NADP-Specifie malate dehydrogenase and glycerate kinase in leaves and evidence for their location in chloroplasts. Biochem. biophys. Res. Commun. 34, 589-593 (1969)
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Hudock, G.A., Fuller, R. C.: Control of triosephosphate dehydrogenase in photosynthesis. Plant Physiol. 40, 1205-1211 (1965) Hurwitz, J., Weissbach, A., Horecker, B. L., Smyrniotis, P. Z. : Spinach phosphoribulokinase. J. biol. Chem. 218, 769-783 (1956) Kachru, R. B., Anderson, L. E. : Inactivation of pea leaf phosphofrucfokinase by light and dithiothreitol. Plant Physiol., in press (1975) Kratz, W. A., Myers, J. : Nutrition and growth of several blue-green algae. Amer. ft. Bot. 42, 282-287 (1955) Ling, K. lt., Paetkan, V., Marcus, R., Lardy, It. A. : Phosphofructokinasc I. Skeletal muscle. In: Methods in enzymology, vol. 9, p. 425429, W. A. Wood, ed. New York: Academic Press 1966 Muto, S., Uritani, I.: Glucose 6-phosphate dehydrogenase from sweet potato. Plant Cell Physiol. 11, 767-776 (1970) Pelroy, R . A . , Bassham, J . A . : Photosynthetic and dark carbon metabolism in unicellular blue-green algae. Arch. Mikrobiol. 86, 25-38 (1972) Pelroy, R. A., Rippka, R., Stanier, R. Y. : The metabolism of glucose by unicellular blue-green algae. Arch. Mikrobiol. 87, 303-322 (1972) Smillie, R. M.: Plant fructose-l,6-diphosphatasc. In: Fructose-l,6-diphosphatase and its role in gluconeogenesis, p. 31-41, McGilvery, R. W., Pogell, B. M., eds. Baltimore: Port City Press 1964 Tabita, F . R . , McFadden, B.A.: Regulation of ribulose-l,5-diphosphate carboxylasc by 6-phospho-D-gluconate. Biochem. biophys. Res. Commun. 48, 1153-1159 (1972) Tabita, F . R . , McFadden, B.A.: D-Ribulose 1,5-diphosphate carboxylase from Rhodospirillum rubrum I Levels, purification, and effects of metallic ions. J. biol. Chem. 249, 3453-3468 (1974) Wu, R., Racker, E. : Regulatory mechanisms in carbohydrate metabolism I I I Limiting factors in glycolysis of ascites tumor cells. J. biol. Chem. 284, 1029-1035 (1959) Ziegler, H., Ziegler, I.: Die lichtindnzierte Aktivit~tssteigerung der NADP+-abh~ngigen Glyccrinaldehyd-3-phosphat-dehydrogenase. V. Das Verhalten yon Mceresalgen. Planta (Berl.) 72, 162-169 (1967) Ziegler, H., Ziegler, I., Beth, K.: Die lichtinduzierte Aktivit~tssteigerung der NADP+abh~ingigen Glyccrinaldehyd-3-Phosphat-Dehydrogenase. VI. Der Einflull des Zellkernes auf den Effekt. Planta (Berl.) 72, 247-251 (1967)
