Plastidial Phosphorylase Research Articles

The Plastidial Glucan Phosphorylase Affects the Maltooligosaccharide Metabolism in Parenchyma Cells of Potato (Solanum tuberosum L.) Tuber Discs.

Maltodextrin metabolism is thought to be involved in both starch initiation and degradation. In this study, potato tuber discs from transgenic lines containing antisense constructs against the plastidial and cytosolic isoforms of α-glucan phosphorylase and phosphoglucomutase were used to evaluate their influences on the conversion of externally supplied glucose-1-phosphate into soluble maltodextrins, as compared to wild-type potato tubers (Solanum tuberosum L. cv. Desiree). Relative maltodextrin amounts analyzed by capillary electrophoresis with laser-induced fluorescence revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30, as opposed to tubers repressing the plastidial glucan phosphorylase. The results presented here support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate-independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucosyl polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.

Carbon pathways during transitory starch degradation in Arabidopsis differentially affect the starch granule number and morphology in the dpe2/phs1 mutant background

The Arabidopsis knockout mutant lacking both the cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) had a dwarf-growth phenotype, a reduced and uneven distribution of starch within the plant rosettes, and a lower starch granule number per chloroplast under standard growth conditions. In contrast, a triple mutant impaired in starch degradation by its additional lack of the glucan, water dikinase (GWD) showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to the wild type. We concluded that ongoing starch degradation is mainly responsible for the observed phenotype of dpe2/phs1. Next, we generated two further triple mutants lacking either the phosphoglucan, water dikinase (PWD), or the disproportionating enzyme 1 (DPE1) in the background of the double mutant. Analysis of the starch metabolism revealed that even minor ongoing starch degradation observed in dpe2/phs1/pwd maintained the double mutant phenotype. In contrast, an additional blockage in the glucose pathway of starch breakdown, as in dpe2/phs1/dpe1, resulted in a nearly starch-free phenotype and massive chloroplast degradation. The characterized mutants were discussed in the context of starch granule formation.

Starch granule initiation in Arabidopsis thaliana chloroplasts.

The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.

The Rice Plastidial Phosphorylase Participates Directly in Both Sink and Source Processes.

The plastidial starch phosphorylase (Pho1) functions in starch metabolism. A distinctive structural feature of the higher Pho1 is a 50-82-amino-acid long peptide (L50-L82), which is absent in phosphorylases from non-plant organisms. To study the function of the rice Pho1 L80 peptide, we complemented a pho1- rice mutant (BMF136) with the wild-type Pho1 gene or with a Pho1 gene lacking the L80 region (Pho1ΔL80). While expression of Pho1 in BMF136 restored normal wild-type phenotype, the introduction of Pho1ΔL80 enhanced the growth rate and plant productivity above wild-type levels. Mass spectrometry analysis of proteins captured by anti-Pho1 showed the surprising presence of PsaC, the terminal electron acceptor/donor subunit of photosystem I (PSI). This unexpected interaction was substantiated by reciprocal immobilized protein pull-down assays of seedling extracts and supported by the presence of Pho1 on isolated PSI complexes resolved by blue-native gels. Spectrophotometric studies showed that Pho1ΔL80 plants exhibited modified PSI and enhanced CO2 assimilation properties. Collectively, these findings indicate that the higher plant Pho1 has dual roles as a potential modulator of source and sink processes.

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5–7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

Characterization of the functional interactions of plastidial starch phosphorylase and starch branching enzymes from rice endosperm during reserve starch biosynthesis

Functional interactions of plastidial phosphorylase (Pho1) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Pho1 and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Pho1. Based on our in vitro data, in the developing rice endosperm, Pho1 appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Pho1. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Pho1 performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Pho1 is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis.

Starch Synthase 4 and Plastidal Phosphorylase Differentially Affect Starch Granule Number and Morphology.

The process of starch granule formation in leaves of Arabidopsis (Arabidopsis thaliana) is obscure. Besides STARCH SYNTHASE4 (SS4), the PLASTIDIAL PHOSPHORYLASE (PHS1) also seems to be involved, since dpe2-1/phs1a double mutants lacking both PHS1 and the cytosolic DISPROPORTIONATING ENZYME2 (DPE2) displayed only one starch granule per chloroplast under normal growth conditions. For further studies, a dpe2-1/phs1a/ss4 triple mutant and various combinations of double mutants were generated and metabolically analyzed with a focus on starch metabolism. The dpe2-1/phs1a/ss4 mutant revealed a massive starch excess phenotype. Furthermore, these plants grown under 12 h of light/12 h of dark harbored a single large and spherical starch granule per plastid. The number of starch granules was constant when the light/dark regime was altered, but this was not observed in the parental lines. With regard to growth, photosynthetic parameters, and metabolic analyses, the triple mutant additionally displayed alterations in comparison with ss4 and dpe2-1/phs1a The results clearly illustrate that PHS1 and SS4 are differently involved in starch granule formation and do not act in series. However, SS4 appears to exert a stronger influence. In connection with the characterized double mutants, we discuss the generation of starch granules and the observed formation of spherical starch granules.

Rice Endosperm Starch Phosphorylase (Pho1) Assembles with Disproportionating Enzyme (Dpe1) to Form a Protein Complex That Enhances Synthesis of Malto-oligosaccharides

Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pulldown experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multienzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger maltooligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30 °C. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly.

Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature

Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Pho1; EC 2.4.1.1) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of α-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Pho1 has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Pho1 activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures.

Double Knockout Mutants of Arabidopsis Grown under Normal Conditions Reveal that the Plastidial Phosphorylase Isozyme Participates in Transitory Starch Metabolism

In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1×phs1a and mex1×phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1×phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-14C]glucose 1-phosphate, [U-14C]sucrose, [U-14C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-14C]sucrose plus unlabelled equimolar glucose 1-phosphate. 14C-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced 14C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-14C]glucose 1-phosphate or adenosine-[U-14C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro 14C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.

The failure to express a protein disulphide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice

The rice somaclonal mutant T3612 produces small grains with a floury endosperm, caused by the loose packing of starch granules. The positional cloning of the mutation revealed a deletion in a gene encoding a protein disulphide isomerase-like enzyme (PDIL1-1). In the wild type, PDIL1-1 was expressed throughout the plant, but most intensely in the developing grain. In T3612, its expression was abolished, resulting in a decrease in the activity of plastidial phosphorylase and pullulanase, and an increase in that of soluble starch synthase I and ADP-glucose pyrophosphorylase. The amylopectin in the T3612 endosperm showed an increase in chains with a degree of polymerization 8–13 compared with the wild type. The expression in the mutant's endosperm of certain endoplasmic reticulum stress-responsive genes was noticeably elevated. PDIL1-1 appears to play an important role in starch synthesis. Its absence is associated with endoplasmic reticulum stress in the endosperm, which is likely to underlie the formation of the floury endosperm in the T3612 mutant.

Glucose-1-Phosphate Transport into Protoplasts and Chloroplasts from Leaves of Arabidopsis

Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.

Glucose 1‐phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules

Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [(14)C]glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

The Relation of Starch Phosphorylases to Starch Metabolism in Wheat

Tissues of wheat (Triticum aestivum L., var. Star) exhibit three starch phosphorylase activity forms resolved by non-denaturing polyacrylamide gel affinity electrophoresis (P1, P2 and P3). Compartmentation analysis of young leaf tissues showed that P3 is plastidic, whereas P1 and P2 are cytosolic. P1 exhibits a strong binding affinity to immobilized glycogen upon electrophoresis, whereas P2 and the chloroplastic P3 do not. Cytosolic leaf phosphorylase was purified to homogeneity by affinity chromatography. The single polypeptide product constituted both the P1 and P2 activity forms. Probes for the detection of phosphorylase transcripts were derived from cDNA sequences of cytosolic and plastidic phosphorylases, and these-together with activity assays and a cytosolic phosphorylase-specific antiserum-were used to monitor phosphorylase expression in leaves and seeds. Mature leaves contained only plastidic phosphorylase, which was also strongly evident in the endosperm of developing seeds at the onset of reserve starch accumulation. Germinating seeds contained only cytosolic phosphorylase, which was restricted to the embryo. Plastidic phosphorylase thus appears to be associated with transitory leaf starch metabolism and with the initiation of seed endosperm reserve starch accumulation, but it plays no role in the degradation of the reserve starch. Cytosolic phosphorylase may be involved in the processing of incoming carbohydrate during rapid tissue growth.

Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress.

To study the role of the plastidial alpha-glucan phosphorylase in starch metabolism in the leaves of Arabidopsis, two independent mutant lines containing T-DNA insertions within the phosphorylase gene were identified. Both insertions eliminate the activity of the plastidial alpha-glucan phosphorylase. Measurement of other enzymes of starch metabolism reveals only minor changes compared with the wild type. The loss of plastidial alpha-glucan phosphorylase does not cause a significant change in the total accumulation of starch during the day or its remobilization at night. Starch structure and composition are unaltered. However, mutant plants display lesions on their leaves that are not seen on wild-type plants, and mesophyll cells bordering the lesions accumulate high levels of starch. Lesion formation is abolished by growing plants under 100% humidity in still air, but subsequent transfer to circulating air with lower humidity causes extensive wilting in the mutant leaves. Wilted sectors die, causing large lesions that are bordered by starch-accumulating cells. Similar lesions are caused by the application of acute salt stress to mature plants. We conclude that plastidial phosphorylase is not required for the degradation of starch, but that it plays a role in the capacity of the leaf lamina to endure a transient water deficit.

Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization.

Higher plants contain two types of phosphorylase (EC 2.4.1.1). One type is plastidic (Phol) and the other resides in the cytosol (Pho2). For Solanum tuberosum L., two highly homologous Pho1-type sequences (designated as Pho1a and Pho1b, respectively) have been described that occur both in a homodimeric, (Pho1a)2, and a heterodimeric, Pho1a-Pho1b, state [U. Sonnewald et al. (1995) Plant Mol Biol 27:567 576; T. Albrecht et al. (1998) Eur J Biochem 251:981-991]. We present a spatial and temporal analysis of the expression patterns of the Pho1-type phosphorylases in S. tuberosum. Expression was analyzed at transcript, protein and activity levels. The specificity of both the probes and the antibodies used was carefully determined to ensure selectivity of detection. For both the Pho1a and Pho1b probes the degree of cross-hybridization was estimated. Peptide scanning identified the epitopes of the anti-Pho 1a and anti-Pho 1b antibodies. Expression of the two Pho1-type genes was analyzed in various organs of the potato plant. In all organs studied the Pho1a transcript levels exceeded those of Pho1b. Furthermore, leaves of a given developmental stage were sampled during the light period and were analyzed for transcript and protein levels and for various carbohydrate pools as well. The data show that in leaves the Pho1a gene expression closely corresponds to starch accumulation, suggesting that the enzyme fulfils a metabolic function within the process of starch biosynthesis. In tubers, Pho1a is constitutively expressed in the parenchyma cells whereas expression of the Pho1b, gene is restricted to cells in close vicinity of the vascular tissue.

Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L.) by exogenously supplied carbohydrates in excised leaf discs

Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L.) by exogenously supplied carbohydrates in excised leaf discs

Light Alters Cytosolic and Plastidic Phosphorylase Distribution in Pearl Millet Leaves.

In pearl millet (Pennisetum americanum) seedlings, although the cytosolic phosphorylase was present in all organs, the plastidic phosphorylase was restricted to the leaf. Intercellular fractionation of the leaf revealed that cytosolic and plastidic phosphorylase were localized in the mesophyll and bundle-sheath cells, respectively. In dark-grown leaves phosphorylase activity increased from the leaf base to the tip. The dark-grown leaves possessed both cytosolic and plastidic phosphorylase isoforms; however, their distribution followed different patterns along the length of the leaf. The plastidic phosphorylase level increased from the base to the tip of the leaf, and the cytosolic phosphorylase level was higher in the basal half of the leaf and declined toward the tip. In light-grown leaves phosphorylase activity was higher in a region near the leaf base and declined toward the leaf tip. Light stimulated cytosolic phosphorylase level and repressed plastidic phosphorylase level, resulting in an altered distribution of the respective phosphorylase isoforms along the length of the light-grown leaf. Since pearl millet leaf possesses a cell maturity and chloroplast development gradient from the leaf base to the tip, the inverse effect of light on cytosolic and plastidic phosphorylase levels might have been determined by its interaction with these gradients.

Polysaccharide Fraction from Higher Plants which Strongly Interacts with the Cytosolic Phosphorylase Isozyme

From leaves of Spinacia oleracea L. or from Pisum sativum L. and from cotyledons of germinating pea seeds a high molecular weight polysaccharide fraction was isolated. The apparent size of the fraction, as determined by gel filtration, was similar to that of dextran blue. Following acid hydrolysis the monomer content of the polysaccharide preparation was studied using high pressure liquid and thin layer chromatography. Glucose, galactose, arabinose, and ribose were the main monosaccharide compounds. The native polysaccharide preparation interacted strongly with the cytosolic isozyme of phosphorylase (EC 2.4.1.1). Interaction with the plastidic phosphorylase isozyme(s) was by far weaker. Interaction with the cytosolic isozyme was demonstrated by affinity electrophoresis, kinetic measurements, and by (14)C-labeling experiments in which the glucosyl transfer from [(14)C]glucose 1-phosphate to the polysaccharide preparation was monitored.