Planta
Planta 143, 63-65 (1978)
,9 by Springer-Verlag 1978
Enzymes of Glycogen Mobilization in the Photosynthetic Procaryote, Anacystis nidulans Martin L e h m a n n and G/inter W 6 b e r Biochemie, Fachbereich Chemic der Philipps-Universit/it, Lahnberge, Postfach 1929, D-3550 Marburg/Lahn, Federal Republic of Germany
Abstract. Glycogen, the principal storage c o m p o u n d of assimilatory p r o d u c t s in Anacystis nidulans, is synthesized in the light and degraded in the dark. 14Clabelled glycogen and its radioactive limit dextrin obtained by phosphorylase action were used as substrates to identify enzymes involved in glycogen mobilization. A crude h o m o g e n a t e of ceils kept in the dark contained the following enzymes: glycogen p h o s p h o r y l a s e (EC 2.4.1.1) that is firmly b o u n d to glycogen, a debranching enzyme that hydrolyzes 1,6~-glucosidic bonds, and an ~-glucosidase (EC 3.2.1.20). Other amylolytic enzymes were not detectable. Using ion exchange c h r o m a t o g r a p h y on DEAE-cellulose, c~-glucosidase and the debranching enzyme could be partly separated f r o m each other and completely f r o m the p h o s p h o r y l a s e - g l y c o g e n complex. On the basis o f their k n o w n substrate specificities, the c o o p e r a t i o n of these 3 enzymes is sufficient to a c c o u n t for the complete conversion of glycogen into glucose and glucose 1-phosphate. Key words: Anacystis - D e b r a n c h i n g enzyme - ~Glucosidase - G l y c o g e n mobilization - Glycogen p h o s p h o r y l a s e - Polysaccharide (reserve).
Introduction c~-Glucan catabolism in p h o t o t r o p h i c organisms is only incompletely understood. In photosynthetic cells of eukaryotic plants, starch phosphorylase is consistently found, and the occurrence o f ~-amylase (EC 3.2.1.1) and fi-amylase (EC 3.2.1.2) is occasionally reported (Haapala, 1969; W a n k a et al., 1970; Peavey et al., 1977). A m o n g the prokaryotes, attention, f r o m an evolutionary point of view, has been given to a-glucan p h o s p h o r y l a s e (Fredrick, 1970) and, in the context o f p h o t o o r g a n o t r o p h i c vs. heterotro-
phic growth, to the blue-green algae (Pelroy et al., 1972). Hydrolases f r o m blue-green algae specific for the cleavage o f 1,4-c~-glucosidic or 1,6-c~-glucosidic bonds have not been reported. Physiological conditions which lead to accumulation or mobilization of glycogen in Anacystis nidulans are similar to those f o u n d in heterotrophic bacteria ( L e h m a n n and W6ber, 1976), and the analogy m a y extend to include the associated enzymes. Of the degradative enzymes in Escherichia coli, glycogen phosphorylase (EC 2.4.1.1, Chert and Segel, 1968) and a debranching enzyme (Jeanningros et al., 1976) have been purified and characterized. As part o f a study of light/dark regulation of glycogen metabolism in A. nidulans, we have tried to identify enzymes that might be involved in glycogen degradation.
Material and Methods Anacystis nidulans, strain 1402-1 from the G6ttingen Collection, was grown continuously in a chemostat under nitrogen-limited conditions (Lehmann and W6ber, 1978a). 200 ml of overflow was stored in the dark for 15 h during which time the intracellular glycogen level decreased by about 50%; cells were then converted into spheroplasts (Lehmann and W6ber, 1978b) and harvested by centrifugation (5 min, 2000 g). The pellet was lysed by resuspension in 5 ml 50 mM tris/maleate buffer, pH 6.2, and again centrifuged (10 rain, 1000 g) to remove membraneous materiaI. The supernatant liquid containing, in addition to soluble enzymes, glycogen and some of the chromoproteins was either used directly as a source of enzymes or adsorbed on DEAE-cellulose. The column (10• 150 mm), washed with 50 mM tris/maleate buffer, pH 6.2, until the phosphorylase-glycogen complex had been removed, was subsequently washed with a 0-0.5 M NaC1 gradient in buffer which eluted 2 different glycosidases. *4C-labelled glycogen was prepared as described previously (Lehmann and W6ber, 1977); l'~C-labelled glycogen phosphorylase limit dextrin was obtained after exhaustive incubation with rabbit muscle phosphorylase purified by hydrophobic chromatography according to Taylor et al., 1975. A test for the identification of
0032-0935/78/0143/0063/$01.00
64
M. L e h m a n n and G. W6ber: Enzymes of Glycogen Mobilization in Anacystis
glycosidases in A. nidulans extracts comprised 0.2 ml radioactive substrate solution (10 mg/ml, 3200 s-z), 0.5 ml extracts and 0.3 ml 300 m M tris/maleate buffer, p H 6.8. After incubation for 2 h at 35 ~ C, proteins were removed by heating and subsequent centrifugation, the supernatant liquid was reduced in vacuo to 0.I ml and subjected to paper chromatography. Reaction products were separated by the multiple-ascent method with n-butanol: pyridin : water (6:4:3, v/v/v) as the solvent (French et al., 1966), detected with a radiochromatogram scanner and identified by comparison with an authentic maltodextrin mixture. c~-Glucosidase (EC 3.2.1.20) was tested for with p-nitrophenyl C~-D-glucoside as the substrate in a digest otherwise as above. After a 0.5 2 h incubation, the reaction was stopped with enough 0.l M NazCO3 solution to bring the p H to 10.0, and the absorption of p-nitrophenolate at 400 n m was determined.
Table 1. Products after degradation of [lr and 14Clabelled phosphorylase limit dextrin by a crude extract of Anacystis nidulans
A B C D
Substrate
Addition a
Products
Glycogen Glycogen Limit dextrin Limit dextrin
Phosphate Phosphate
n.d. b
G., G 4, G1, G-I-P c'~ G., G 4, G~ G,, G4, G 1
Sodium phosphate buffer, pH 6.5, 10 m M final concentration b Not detectable ~ G~, G4, Gn denote respectively glucose, maltotetraose and larger h o m o l o g o u s oligosaccharides; G-I-P, glucose 1-phosphate a Aliquots of incubations B and D were treated, in addition, with alkaline phosphatase. By comparison with the untreated sample, glucose 1-phosphate could be determined as glucose
Results
Radioactive products identified after incubation of labelled polysaccharides under various conditions with a crude extract of Anacystis nidulans are shown in Table 1. Results of experiments A and B indicate that phosphorolytic attack must precede any hydrolytic step, since only in exp. B were maltosaccharides found. The appearance of both maltosaccharides and glucose was the result of 2 distinct enzyme activities, because glycogen phosphorylase limit dextrin, when degraded with authentic bacterial isoamylase (EC 3.2.I.68), produced only maltotetraose and higher maltosaccharides (control not shown). On the other hand, phosphorylase limit dextrin was a suitable substrate for hydrolases (exp. D). To further characterize glycogen-degrading enzymes in A. nidulans, the crude cell extract was partially purified by chromatography on DEAE-cellulose. An initial wash, the 0-0.5 M NaC1 and the 0.5-1 M NaC1 eluates were collected separately and monitored for the same activities as before. The 0.5-1 M NaC1 eluate did not contain any amylolytic activity. Results of tests with the other fractions are listed in Table 2. Again, native glycogen was an inert substrate (exp. E), and glycogen phosphorylase, free of hydrolytic enzymes, was eluted from the column by the initial wash (exp. F). Analysis of products produced in exp. G and H leads to the conclusion that phosphorylase limit dextrin is a substrate for a debranching enzyme. This debranching enzyme hydrolyzes 1,6-c~-glucosidic linkages thereby liberating linear maltosaccharides, the smallest of which is maltotetraose. This evidence and the lack of further phosphorolysis (exp. H) are proof of the near-ideal structure of the limit-dextrin substrate (Lee and Whelan, 1971). It can be concluded, then, that a direct debranching enzyme occurs in A. nidulans as in other bacteria and plants and, secondly, that its substrate specificity may be similar to that
Table 2. Products after degradation of [14C]glycogen and 14Clabelled phosphorylase limit dextrin by a partially purified extract of Anacystis nidulans Substrate
Addition a
E F G H
Glycogen Glycogen Limit dextrin Limit dextrin
Phosphate Phosphate
a b ~ for
Sodium phosphate buffer, Abbreviations as in Table Each sample was passed desalting before subjection
Enzyme activity in Products buffer wash
0-0.5 M NaCI
+
+ c + e
-
n.d. b G- 1-Pb On, G4, G1 b On, G4, GI
pH 6.5, l0 m M final concentration 1 over Biogel P-2 (Pasteur pipette) to paper chromatography
of the Escherichia coli enzyme. Unlike other bacterial isoamylases, this debranching enzyme has a high affinity for glycogen phosphorylase limit dextrin and low affinity for native glycogen (Jeanningros et al., 1976). Whether this is merely an adaptive phenomenon of enzymes operating closely in sequence or has any regulatory significance remains to be seen. No evidence for the occurrence of a pullulanase (EC 3.2.1.41) was found. Free glucose was apparently formed from maltosaccharides in a subsequent reaction catalyzed by c~glucosidase. The presence of the enzyme was shown independently by the hydrolysis of p-nitrophenyl C~-Dglucoside. The absence of maltose and maltotriose among the reaction products precludes the occurrence of both s-amylase and/?-amylase. The activities of glycogen phosphorylase, debranching enzyme and ~-glucosidase after column chromatography were 167, 0.83 and 0.67 nkat/mg protein respectively. We have calculated that the activity of debranching enzyme and ~-glucosidase mea-
M. Lehmann and G. W6ber: Enzymes of Glycogen Mobilization in Anacystis
sured after a purification step is at least 10 times higher than would be necessary to account for the rate of glycogen degradation in vivo during a dark period. Figures of enzyme activities for crude cell homogenates which would be more relevant to in vivo rates of glycogen degradation are not available because of the presence in crude extracts of coloured substances interfering with the quantitative assay. In conclusion, we believe to have presented sufficient evidence for the presence of glycogen phosphorylase, debranching enzyme, and a-glucosidase and, in addition, demonstrated the absence of other enzymes conceivably involved in glycogen degratation. These 3 enzymes have a substrate specificity such that through sequential operation they can account for the complete conversion of glycogen into glucose and glucose 1-phosphate. This work was supported by a grant (SFB 103/A 5) from the Deutsche Forschungsgemeinschaft.
References Chen, G.S., Segel, L.H.: Purification and properties of glycogen phosphorylase from Escherichia eoli. Arch. Biochem. Biophys. 127, 175-186 (1968) Fredrick, J.F.: Evolution of polyglucoside-synthesizing isozymes in the algae. In: Phylogenesis and morphogenesis in the algae, Fredrick, J.F., Klein, R.M., (eds.) Vo]. 1975, pp. 524-530. Ann. New York Acad. Sci. New York: Academy 1970 French, D., Pulley, A.P., Abdullah, M., Linden, J.C. : Two-dimensional paper chromatography interspersed with reaction on the paper. J. Chromatog. 24, 271-276 (1966)
65
Haapala, H. : Studies on the activity of beta-amylase in the chloroplasts of Stellaria media during prolonged illumination. Physiol. Plant. 22, 140-146 (1969) Jeanningros, R., Creuzet-Sigal, N., Frixon, C., Cattan6o, J. : Purification and properties of a debranching enzyme from Escherichia cola Biochim. Biophys. Acta 438, 186-199 (1976) Lee, E.Y.C., Whelan, W.J.: Glycogen and starch-debranching enzymes, In: The enzymes, Boyer, P.D., (ed.) Vol. 5, pp. 191-234, 3rd ed. New York: Academic Press 1971 Lehmann, M., W6ber, G. : Accumulation, mobilization and turnover of glycogen in the blue-green bacterium, Anacystis nidulans. Arch. Microbiol. 111, 93-97 (1976) Lehmann, M., W6ber, G. : [U-14C]-labelled glycogen, maltodextrin mixture, maltose and glucose : Preparation by photoassimilation of 1~CO2 in Anacystis nidulans and by combined affinity chromatography and selective enzymic degradation. Carbohydr. Res. 56, 357 362 (1977) Lehmann, M., W6ber, G.: Continuous culture in a chemostat of the photosynthetic prokaryote, Anacystis nidulans, under nitrogen-limiting conditions. Mol. Cell. Biochem. 19, 155-163 (1978a) Lehmann, M., W6ber, G. : Light modulation of glycogen phosphorylase activity in the blue-green bacterium, Anacystis nidulans. Plant. Cell. Environ. 1, 155-160 (1978b) Peavey, D.G., Steup, M., Gibbs, M.: Characterization of starch breakdown in the intact spinach chloroplast. Plant Physiol. 60, 305-308 (1977) Pelroy, R.A., Rippka, R., Stanier, R.Y.: Metabolism of glucose by unicellular blue-green algae. Arch. Mikrobiol. 87, 303-322 (1972) Taylor, C., Cox, A.J., Kernohan, J.C., Cohen, P.: Debranching enzyme from rabbit skeletal muscle. Europ. J. Biochem. 51, 105-115 (1975) Wanka, F., Joppen, M.M.J., Kuyper, C.M.A.: Starch-degrading enzymes in synchronous cultures of Chlorella. Z. Pflanzenphysiol. 62, 146 157 (1970) Received 16 May; accepted 5 July 1978
Planta 143, 63-65 (1978)
,9 by Springer-Verlag 1978
Enzymes of Glycogen Mobilization in the Photosynthetic Procaryote, Anacystis nidulans Martin L e h m a n n and G/inter W 6 b e r Biochemie, Fachbereich Chemic der Philipps-Universit/it, Lahnberge, Postfach 1929, D-3550 Marburg/Lahn, Federal Republic of Germany
Abstract. Glycogen, the principal storage c o m p o u n d of assimilatory p r o d u c t s in Anacystis nidulans, is synthesized in the light and degraded in the dark. 14Clabelled glycogen and its radioactive limit dextrin obtained by phosphorylase action were used as substrates to identify enzymes involved in glycogen mobilization. A crude h o m o g e n a t e of ceils kept in the dark contained the following enzymes: glycogen p h o s p h o r y l a s e (EC 2.4.1.1) that is firmly b o u n d to glycogen, a debranching enzyme that hydrolyzes 1,6~-glucosidic bonds, and an ~-glucosidase (EC 3.2.1.20). Other amylolytic enzymes were not detectable. Using ion exchange c h r o m a t o g r a p h y on DEAE-cellulose, c~-glucosidase and the debranching enzyme could be partly separated f r o m each other and completely f r o m the p h o s p h o r y l a s e - g l y c o g e n complex. On the basis o f their k n o w n substrate specificities, the c o o p e r a t i o n of these 3 enzymes is sufficient to a c c o u n t for the complete conversion of glycogen into glucose and glucose 1-phosphate. Key words: Anacystis - D e b r a n c h i n g enzyme - ~Glucosidase - G l y c o g e n mobilization - Glycogen p h o s p h o r y l a s e - Polysaccharide (reserve).
Introduction c~-Glucan catabolism in p h o t o t r o p h i c organisms is only incompletely understood. In photosynthetic cells of eukaryotic plants, starch phosphorylase is consistently found, and the occurrence o f ~-amylase (EC 3.2.1.1) and fi-amylase (EC 3.2.1.2) is occasionally reported (Haapala, 1969; W a n k a et al., 1970; Peavey et al., 1977). A m o n g the prokaryotes, attention, f r o m an evolutionary point of view, has been given to a-glucan p h o s p h o r y l a s e (Fredrick, 1970) and, in the context o f p h o t o o r g a n o t r o p h i c vs. heterotro-
phic growth, to the blue-green algae (Pelroy et al., 1972). Hydrolases f r o m blue-green algae specific for the cleavage o f 1,4-c~-glucosidic or 1,6-c~-glucosidic bonds have not been reported. Physiological conditions which lead to accumulation or mobilization of glycogen in Anacystis nidulans are similar to those f o u n d in heterotrophic bacteria ( L e h m a n n and W6ber, 1976), and the analogy m a y extend to include the associated enzymes. Of the degradative enzymes in Escherichia coli, glycogen phosphorylase (EC 2.4.1.1, Chert and Segel, 1968) and a debranching enzyme (Jeanningros et al., 1976) have been purified and characterized. As part o f a study of light/dark regulation of glycogen metabolism in A. nidulans, we have tried to identify enzymes that might be involved in glycogen degradation.
Material and Methods Anacystis nidulans, strain 1402-1 from the G6ttingen Collection, was grown continuously in a chemostat under nitrogen-limited conditions (Lehmann and W6ber, 1978a). 200 ml of overflow was stored in the dark for 15 h during which time the intracellular glycogen level decreased by about 50%; cells were then converted into spheroplasts (Lehmann and W6ber, 1978b) and harvested by centrifugation (5 min, 2000 g). The pellet was lysed by resuspension in 5 ml 50 mM tris/maleate buffer, pH 6.2, and again centrifuged (10 rain, 1000 g) to remove membraneous materiaI. The supernatant liquid containing, in addition to soluble enzymes, glycogen and some of the chromoproteins was either used directly as a source of enzymes or adsorbed on DEAE-cellulose. The column (10• 150 mm), washed with 50 mM tris/maleate buffer, pH 6.2, until the phosphorylase-glycogen complex had been removed, was subsequently washed with a 0-0.5 M NaC1 gradient in buffer which eluted 2 different glycosidases. *4C-labelled glycogen was prepared as described previously (Lehmann and W6ber, 1977); l'~C-labelled glycogen phosphorylase limit dextrin was obtained after exhaustive incubation with rabbit muscle phosphorylase purified by hydrophobic chromatography according to Taylor et al., 1975. A test for the identification of
0032-0935/78/0143/0063/$01.00
64
M. L e h m a n n and G. W6ber: Enzymes of Glycogen Mobilization in Anacystis
glycosidases in A. nidulans extracts comprised 0.2 ml radioactive substrate solution (10 mg/ml, 3200 s-z), 0.5 ml extracts and 0.3 ml 300 m M tris/maleate buffer, p H 6.8. After incubation for 2 h at 35 ~ C, proteins were removed by heating and subsequent centrifugation, the supernatant liquid was reduced in vacuo to 0.I ml and subjected to paper chromatography. Reaction products were separated by the multiple-ascent method with n-butanol: pyridin : water (6:4:3, v/v/v) as the solvent (French et al., 1966), detected with a radiochromatogram scanner and identified by comparison with an authentic maltodextrin mixture. c~-Glucosidase (EC 3.2.1.20) was tested for with p-nitrophenyl C~-D-glucoside as the substrate in a digest otherwise as above. After a 0.5 2 h incubation, the reaction was stopped with enough 0.l M NazCO3 solution to bring the p H to 10.0, and the absorption of p-nitrophenolate at 400 n m was determined.
Table 1. Products after degradation of [lr and 14Clabelled phosphorylase limit dextrin by a crude extract of Anacystis nidulans
A B C D
Substrate
Addition a
Products
Glycogen Glycogen Limit dextrin Limit dextrin
Phosphate Phosphate
n.d. b
G., G 4, G1, G-I-P c'~ G., G 4, G~ G,, G4, G 1
Sodium phosphate buffer, pH 6.5, 10 m M final concentration b Not detectable ~ G~, G4, Gn denote respectively glucose, maltotetraose and larger h o m o l o g o u s oligosaccharides; G-I-P, glucose 1-phosphate a Aliquots of incubations B and D were treated, in addition, with alkaline phosphatase. By comparison with the untreated sample, glucose 1-phosphate could be determined as glucose
Results
Radioactive products identified after incubation of labelled polysaccharides under various conditions with a crude extract of Anacystis nidulans are shown in Table 1. Results of experiments A and B indicate that phosphorolytic attack must precede any hydrolytic step, since only in exp. B were maltosaccharides found. The appearance of both maltosaccharides and glucose was the result of 2 distinct enzyme activities, because glycogen phosphorylase limit dextrin, when degraded with authentic bacterial isoamylase (EC 3.2.I.68), produced only maltotetraose and higher maltosaccharides (control not shown). On the other hand, phosphorylase limit dextrin was a suitable substrate for hydrolases (exp. D). To further characterize glycogen-degrading enzymes in A. nidulans, the crude cell extract was partially purified by chromatography on DEAE-cellulose. An initial wash, the 0-0.5 M NaC1 and the 0.5-1 M NaC1 eluates were collected separately and monitored for the same activities as before. The 0.5-1 M NaC1 eluate did not contain any amylolytic activity. Results of tests with the other fractions are listed in Table 2. Again, native glycogen was an inert substrate (exp. E), and glycogen phosphorylase, free of hydrolytic enzymes, was eluted from the column by the initial wash (exp. F). Analysis of products produced in exp. G and H leads to the conclusion that phosphorylase limit dextrin is a substrate for a debranching enzyme. This debranching enzyme hydrolyzes 1,6-c~-glucosidic linkages thereby liberating linear maltosaccharides, the smallest of which is maltotetraose. This evidence and the lack of further phosphorolysis (exp. H) are proof of the near-ideal structure of the limit-dextrin substrate (Lee and Whelan, 1971). It can be concluded, then, that a direct debranching enzyme occurs in A. nidulans as in other bacteria and plants and, secondly, that its substrate specificity may be similar to that
Table 2. Products after degradation of [14C]glycogen and 14Clabelled phosphorylase limit dextrin by a partially purified extract of Anacystis nidulans Substrate
Addition a
E F G H
Glycogen Glycogen Limit dextrin Limit dextrin
Phosphate Phosphate
a b ~ for
Sodium phosphate buffer, Abbreviations as in Table Each sample was passed desalting before subjection
Enzyme activity in Products buffer wash
0-0.5 M NaCI
+
+ c + e
-
n.d. b G- 1-Pb On, G4, G1 b On, G4, GI
pH 6.5, l0 m M final concentration 1 over Biogel P-2 (Pasteur pipette) to paper chromatography
of the Escherichia coli enzyme. Unlike other bacterial isoamylases, this debranching enzyme has a high affinity for glycogen phosphorylase limit dextrin and low affinity for native glycogen (Jeanningros et al., 1976). Whether this is merely an adaptive phenomenon of enzymes operating closely in sequence or has any regulatory significance remains to be seen. No evidence for the occurrence of a pullulanase (EC 3.2.1.41) was found. Free glucose was apparently formed from maltosaccharides in a subsequent reaction catalyzed by c~glucosidase. The presence of the enzyme was shown independently by the hydrolysis of p-nitrophenyl C~-Dglucoside. The absence of maltose and maltotriose among the reaction products precludes the occurrence of both s-amylase and/?-amylase. The activities of glycogen phosphorylase, debranching enzyme and ~-glucosidase after column chromatography were 167, 0.83 and 0.67 nkat/mg protein respectively. We have calculated that the activity of debranching enzyme and ~-glucosidase mea-
M. Lehmann and G. W6ber: Enzymes of Glycogen Mobilization in Anacystis
sured after a purification step is at least 10 times higher than would be necessary to account for the rate of glycogen degradation in vivo during a dark period. Figures of enzyme activities for crude cell homogenates which would be more relevant to in vivo rates of glycogen degradation are not available because of the presence in crude extracts of coloured substances interfering with the quantitative assay. In conclusion, we believe to have presented sufficient evidence for the presence of glycogen phosphorylase, debranching enzyme, and a-glucosidase and, in addition, demonstrated the absence of other enzymes conceivably involved in glycogen degratation. These 3 enzymes have a substrate specificity such that through sequential operation they can account for the complete conversion of glycogen into glucose and glucose 1-phosphate. This work was supported by a grant (SFB 103/A 5) from the Deutsche Forschungsgemeinschaft.
References Chen, G.S., Segel, L.H.: Purification and properties of glycogen phosphorylase from Escherichia eoli. Arch. Biochem. Biophys. 127, 175-186 (1968) Fredrick, J.F.: Evolution of polyglucoside-synthesizing isozymes in the algae. In: Phylogenesis and morphogenesis in the algae, Fredrick, J.F., Klein, R.M., (eds.) Vo]. 1975, pp. 524-530. Ann. New York Acad. Sci. New York: Academy 1970 French, D., Pulley, A.P., Abdullah, M., Linden, J.C. : Two-dimensional paper chromatography interspersed with reaction on the paper. J. Chromatog. 24, 271-276 (1966)
65
Haapala, H. : Studies on the activity of beta-amylase in the chloroplasts of Stellaria media during prolonged illumination. Physiol. Plant. 22, 140-146 (1969) Jeanningros, R., Creuzet-Sigal, N., Frixon, C., Cattan6o, J. : Purification and properties of a debranching enzyme from Escherichia cola Biochim. Biophys. Acta 438, 186-199 (1976) Lee, E.Y.C., Whelan, W.J.: Glycogen and starch-debranching enzymes, In: The enzymes, Boyer, P.D., (ed.) Vol. 5, pp. 191-234, 3rd ed. New York: Academic Press 1971 Lehmann, M., W6ber, G. : Accumulation, mobilization and turnover of glycogen in the blue-green bacterium, Anacystis nidulans. Arch. Microbiol. 111, 93-97 (1976) Lehmann, M., W6ber, G. : [U-14C]-labelled glycogen, maltodextrin mixture, maltose and glucose : Preparation by photoassimilation of 1~CO2 in Anacystis nidulans and by combined affinity chromatography and selective enzymic degradation. Carbohydr. Res. 56, 357 362 (1977) Lehmann, M., W6ber, G.: Continuous culture in a chemostat of the photosynthetic prokaryote, Anacystis nidulans, under nitrogen-limiting conditions. Mol. Cell. Biochem. 19, 155-163 (1978a) Lehmann, M., W6ber, G. : Light modulation of glycogen phosphorylase activity in the blue-green bacterium, Anacystis nidulans. Plant. Cell. Environ. 1, 155-160 (1978b) Peavey, D.G., Steup, M., Gibbs, M.: Characterization of starch breakdown in the intact spinach chloroplast. Plant Physiol. 60, 305-308 (1977) Pelroy, R.A., Rippka, R., Stanier, R.Y.: Metabolism of glucose by unicellular blue-green algae. Arch. Mikrobiol. 87, 303-322 (1972) Taylor, C., Cox, A.J., Kernohan, J.C., Cohen, P.: Debranching enzyme from rabbit skeletal muscle. Europ. J. Biochem. 51, 105-115 (1975) Wanka, F., Joppen, M.M.J., Kuyper, C.M.A.: Starch-degrading enzymes in synchronous cultures of Chlorella. Z. Pflanzenphysiol. 62, 146 157 (1970) Received 16 May; accepted 5 July 1978
