Peptide patterns and immunological properties of the cytoplasmic and chloroplastic α-1,4-glucan p... more Peptide patterns and immunological properties of the cytoplasmic and chloroplastic α-1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves have been studied and were compared with those of phosphorylases from other sources. The two spinach leaf phosphorylases were immunologically different; a limited cross-reactivity was observed only at high antigen or antibody concentrations. Peptide mapping of the two enzymes resulted in complex patterns composed of more than 20 fragments; but no peptide was electrophoretically identical in both proteins. Approximately 13 to 15 of the fragments exhibited antigeneity but no cross-reactivity of any peptide was observed. Therefore, the two compartment-specific phosphorylase forms from spinach leaves represent isoenzymes possessing different primary structures. Peptide patterns of potato tuber and rabbit muscle phosphorylase were different from those of the two spinach leaf enzymes. Although the potato tuber phosphorylase resides in the plastidic...
Starch is the major storage carbohydrate in higher plants and of considerable importance for the ... more Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.
The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spi... more The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spinach chloroplast are investigated. During CO 2 fixation in the light increasing levels of Pi (01.0 mM), which have little effect on the rate of fixation, prolong the lag of starch synthesis and ...
In chloroplasts, both biosynthesis and degradation of starch are strictly regulated but the mecha... more In chloroplasts, both biosynthesis and degradation of starch are strictly regulated but the mechanisms involved are still incompletely understood. Recent studies revealed two novel and regulatory relevant aspects in the biochemistry of starch: the phosphorylation of starch and the starch-related metabolism of cytosolic heteroglycans. Starch phosphorylation occurs by a sequential action of two plastidial enzymes, the glucan, water dikinase (GWD; EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD; EC 2.7.9.5). Both enzymes utilize ATP as dual phosphate donor and transfer the terminal phosphate group to water whereas the beta-phosphate is used for esterification of glucosyl moieties. The metabolism of starch-derived degradation products is closely linked to recently discovered cytosolic heteroglycans that possess, as prominent constituents, arabinose, galactose, glucose and fucose. The pattern of glycosidic linkages is highly complex comprising more than 25 different bonds. During the dark period the size distribution or the amount of the cytosolic heteroglycans increases depending on the plant species. As revealed by in vitro 14C labeling assays, the heteroglycans act as both glucosyl acceptors and donors for two cytosolic glucosyl transferases, the phosphorylase (EC 2.4.1.1) and the transglucosidase (EC 2.4.1.25) and, at least in part, both enzymes utilize the same glucosyl acceptor and donor sites. In mutants of Arabidopsis thaliana L. that are deficient in the cytosolic transglucosidase both the structure and (bio)chemical properties of the heteroglycans are altered.
To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants... more To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.
The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants d... more The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70% and less than 5%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a <10 kDa and a >10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived >10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II.
Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrys... more Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephospho...
The oligosaccharides of the Fe(II1)-Zn(I1) purple acid phosphatase of the red kidney bean Determi... more The oligosaccharides of the Fe(II1)-Zn(I1) purple acid phosphatase of the red kidney bean Determination of the structure by a combination of matrix-assisted laser desorptiodionization mass spectrometry and selective enzymic degradation
The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated ... more The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and e-amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by a-amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.
The glucan specifity of the purified chloroplast and non-chloroplast forms of c~-l,4-glucan phosp... more The glucan specifity of the purified chloroplast and non-chloroplast forms of c~-l,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves (Steup and E. Latzko (1979), Planta 145, 69-75) was investigated. Phosphorolysis by the two enzymes was studied using a series of linear maltodextrins (degree of polymerization < 11), amylose, amylopectin, starch, and glycogen as substrates. For all unbranched glucans (amylose and maltodextrins Gs-G11), the chloroplast phosphorylase had a 7-10-fold higher apparent affinity (determined by initial velocity measurements) than the non-chloroplast phosphorylase form. For both enzyme forms, the minimum chain length'~re'~luired for a significant rate of phosphorolysis was five glucose units. Likewise, phosphorolysis ceased when the maltodextrin was converted to maltotetraose. With the chloroplast phosphorylase, maltotetraose was a linear competitive inhibitor with respect to amylose or starch (Ki=0.1 mmol 1-1); the inhibition by maltotetraose was less pronounced with the non-chloroplast enzyme. In contrast to unbranched glucans, the non-chloroplast phosphorylase exhibited a 40-, 50-, and 300-fold higher apparent affinity for amylopectin, starch, and glycogen, respectively, than the chloroplast enzyme. With respect to these kinetic properties the chloroplast phosphorylase resembled the type of ~ maltodextrin phosphorylase".
Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts w... more Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per rain per mg protein was found, whereas a lower value (6 nmol per rain per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per rain per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per rain per mg protein) and contained two of these three phosphorylase forms.
The effect of blue and red light on the synthesis of transfer and 5S ribosomal RNA in autotrophic... more The effect of blue and red light on the synthesis of transfer and 5S ribosomal RNA in autotrophic cultures of Chlorella pyrenoidosa was studied by pulse labeling experiments with tritiated guanosine. Compared with darkness or red light (679 nm), blue light (457 nm) of low intensities (quantum flux: 0.5-5 • 10-,o mol photons cm-2 s-i) stimulated incorporation of guanosine into transfer and 5S ribosomal RNA within the first 5 min of illumination. This blue light effect was abolished by cycloheximide, an inhibitor of protein synthesis on 80S ribosomes, but not by rifampicin, an inhibitor ofchloroplastic transcription, nor by lincomycin, an inhibitor of chloroplastic translation. The rifampicin-insensitive synthesis of transfer and 5S ribosomal RNA was nuclear transcription as shown by RNA-DNA hybridization. The blue light effect on nuclear RNA synthesis was not inhibited by 3-(3,4-dichlorophenyl)-l,l-dimethylurea, an inhibitor of photosynthesis electron transport. Further evidence for a photosynthesis-independent photocontrol of RNA synthesis was provided by experiments with the colorless mutant 125a of Chlorella vulgaris. Blue light stimulated incorporation of guanosine into cytoplasmic 25S and 18S ribosomal RNA as well as into transfer and 5S ribosomal RNA, whereas incorporation in red light was the same as that of the dark control.
A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia ole... more A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia oleracea L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other plants, animals and prokaryotes. Escherichia coli cells carrying the entire cDNA for Pho1 expressed an active phosphorylase, which resembled the properties of the plastidic isozyme of spinach with respect to its low affinity to glycogen. Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity. Plastidic phosphorylase was transcribed in flowers and leaves, but the highest Pho1 transcript levels were found in mature fruits/seeds. This is in agreement with the enzyme activity levels, as Pho1 activity was detected in all tissues tested, but the highest activity was also present in mature fruits/seeds. Since developing seeds are strong sink organs, which import sucrose and accumulate starch, this observation may indicate that plastidic phosphorylase plays a role in starch formation. The assumption has been tested further by a series of induction experiments in which leaf discs from spinach and potato plants were incubated with various carbohydrates. Following incubation, phosphorylase steady-state transcript levels as well as levels of neutral sugars and starch were determined. A similar induction behaviour was found for Pho1 from spinach and Pho1a from potato, indicating the presence of related sugar signal transduction pathways in these two species. In addition, the expression of Pho1a and Agp4 (the large submit of ADPglucose synthase) from potato seems to be partly coordinately regulated by carbohydrates. These data may suggest that the regulation of Pho1 expression is linked to the carbohydrate status of the respective tissue.
Peptide patterns and immunological properties of the cytoplasmic and chloroplastic α-1,4-glucan p... more Peptide patterns and immunological properties of the cytoplasmic and chloroplastic α-1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves have been studied and were compared with those of phosphorylases from other sources. The two spinach leaf phosphorylases were immunologically different; a limited cross-reactivity was observed only at high antigen or antibody concentrations. Peptide mapping of the two enzymes resulted in complex patterns composed of more than 20 fragments; but no peptide was electrophoretically identical in both proteins. Approximately 13 to 15 of the fragments exhibited antigeneity but no cross-reactivity of any peptide was observed. Therefore, the two compartment-specific phosphorylase forms from spinach leaves represent isoenzymes possessing different primary structures. Peptide patterns of potato tuber and rabbit muscle phosphorylase were different from those of the two spinach leaf enzymes. Although the potato tuber phosphorylase resides in the plastidic...
Starch is the major storage carbohydrate in higher plants and of considerable importance for the ... more Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.
The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spi... more The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spinach chloroplast are investigated. During CO 2 fixation in the light increasing levels of Pi (01.0 mM), which have little effect on the rate of fixation, prolong the lag of starch synthesis and ...
In chloroplasts, both biosynthesis and degradation of starch are strictly regulated but the mecha... more In chloroplasts, both biosynthesis and degradation of starch are strictly regulated but the mechanisms involved are still incompletely understood. Recent studies revealed two novel and regulatory relevant aspects in the biochemistry of starch: the phosphorylation of starch and the starch-related metabolism of cytosolic heteroglycans. Starch phosphorylation occurs by a sequential action of two plastidial enzymes, the glucan, water dikinase (GWD; EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD; EC 2.7.9.5). Both enzymes utilize ATP as dual phosphate donor and transfer the terminal phosphate group to water whereas the beta-phosphate is used for esterification of glucosyl moieties. The metabolism of starch-derived degradation products is closely linked to recently discovered cytosolic heteroglycans that possess, as prominent constituents, arabinose, galactose, glucose and fucose. The pattern of glycosidic linkages is highly complex comprising more than 25 different bonds. During the dark period the size distribution or the amount of the cytosolic heteroglycans increases depending on the plant species. As revealed by in vitro 14C labeling assays, the heteroglycans act as both glucosyl acceptors and donors for two cytosolic glucosyl transferases, the phosphorylase (EC 2.4.1.1) and the transglucosidase (EC 2.4.1.25) and, at least in part, both enzymes utilize the same glucosyl acceptor and donor sites. In mutants of Arabidopsis thaliana L. that are deficient in the cytosolic transglucosidase both the structure and (bio)chemical properties of the heteroglycans are altered.
To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants... more To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.
The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants d... more The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70% and less than 5%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10 kDa and a &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II.
Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrys... more Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephospho...
The oligosaccharides of the Fe(II1)-Zn(I1) purple acid phosphatase of the red kidney bean Determi... more The oligosaccharides of the Fe(II1)-Zn(I1) purple acid phosphatase of the red kidney bean Determination of the structure by a combination of matrix-assisted laser desorptiodionization mass spectrometry and selective enzymic degradation
The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated ... more The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and e-amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by a-amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.
The glucan specifity of the purified chloroplast and non-chloroplast forms of c~-l,4-glucan phosp... more The glucan specifity of the purified chloroplast and non-chloroplast forms of c~-l,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves (Steup and E. Latzko (1979), Planta 145, 69-75) was investigated. Phosphorolysis by the two enzymes was studied using a series of linear maltodextrins (degree of polymerization < 11), amylose, amylopectin, starch, and glycogen as substrates. For all unbranched glucans (amylose and maltodextrins Gs-G11), the chloroplast phosphorylase had a 7-10-fold higher apparent affinity (determined by initial velocity measurements) than the non-chloroplast phosphorylase form. For both enzyme forms, the minimum chain length'~re'~luired for a significant rate of phosphorolysis was five glucose units. Likewise, phosphorolysis ceased when the maltodextrin was converted to maltotetraose. With the chloroplast phosphorylase, maltotetraose was a linear competitive inhibitor with respect to amylose or starch (Ki=0.1 mmol 1-1); the inhibition by maltotetraose was less pronounced with the non-chloroplast enzyme. In contrast to unbranched glucans, the non-chloroplast phosphorylase exhibited a 40-, 50-, and 300-fold higher apparent affinity for amylopectin, starch, and glycogen, respectively, than the chloroplast enzyme. With respect to these kinetic properties the chloroplast phosphorylase resembled the type of ~ maltodextrin phosphorylase".
Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts w... more Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per rain per mg protein was found, whereas a lower value (6 nmol per rain per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per rain per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per rain per mg protein) and contained two of these three phosphorylase forms.
The effect of blue and red light on the synthesis of transfer and 5S ribosomal RNA in autotrophic... more The effect of blue and red light on the synthesis of transfer and 5S ribosomal RNA in autotrophic cultures of Chlorella pyrenoidosa was studied by pulse labeling experiments with tritiated guanosine. Compared with darkness or red light (679 nm), blue light (457 nm) of low intensities (quantum flux: 0.5-5 • 10-,o mol photons cm-2 s-i) stimulated incorporation of guanosine into transfer and 5S ribosomal RNA within the first 5 min of illumination. This blue light effect was abolished by cycloheximide, an inhibitor of protein synthesis on 80S ribosomes, but not by rifampicin, an inhibitor ofchloroplastic transcription, nor by lincomycin, an inhibitor of chloroplastic translation. The rifampicin-insensitive synthesis of transfer and 5S ribosomal RNA was nuclear transcription as shown by RNA-DNA hybridization. The blue light effect on nuclear RNA synthesis was not inhibited by 3-(3,4-dichlorophenyl)-l,l-dimethylurea, an inhibitor of photosynthesis electron transport. Further evidence for a photosynthesis-independent photocontrol of RNA synthesis was provided by experiments with the colorless mutant 125a of Chlorella vulgaris. Blue light stimulated incorporation of guanosine into cytoplasmic 25S and 18S ribosomal RNA as well as into transfer and 5S ribosomal RNA, whereas incorporation in red light was the same as that of the dark control.
A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia ole... more A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia oleracea L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other plants, animals and prokaryotes. Escherichia coli cells carrying the entire cDNA for Pho1 expressed an active phosphorylase, which resembled the properties of the plastidic isozyme of spinach with respect to its low affinity to glycogen. Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity. Plastidic phosphorylase was transcribed in flowers and leaves, but the highest Pho1 transcript levels were found in mature fruits/seeds. This is in agreement with the enzyme activity levels, as Pho1 activity was detected in all tissues tested, but the highest activity was also present in mature fruits/seeds. Since developing seeds are strong sink organs, which import sucrose and accumulate starch, this observation may indicate that plastidic phosphorylase plays a role in starch formation. The assumption has been tested further by a series of induction experiments in which leaf discs from spinach and potato plants were incubated with various carbohydrates. Following incubation, phosphorylase steady-state transcript levels as well as levels of neutral sugars and starch were determined. A similar induction behaviour was found for Pho1 from spinach and Pho1a from potato, indicating the presence of related sugar signal transduction pathways in these two species. In addition, the expression of Pho1a and Agp4 (the large submit of ADPglucose synthase) from potato seems to be partly coordinately regulated by carbohydrates. These data may suggest that the regulation of Pho1 expression is linked to the carbohydrate status of the respective tissue.
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Papers by M. Steup