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  • 1
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Analytical chemistry 63 (1991), S. 1463-1466 
    ISSN: 1520-6882
    Source: ACS Legacy Archives
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In turions of Spirodela polyrhiza (L.) Schleiden, net degradation of storage starch is controlled by a special low fluence response of phytochrome requiring illumination for several days. This light effect has been used to study protein–starch interactions that occur prior to and during net degradation of starch. Following various pretreatments on S. polyrhiza turions, native starch granules were isolated and two fractions of starch-related proteins were distinguished: proteins enclosed within the starch particles (starch-internalized proteins) and those attached to the surface (starch-associated proteins). The pattern of starch-associated proteins as resolved by SDS-PAGE was more complex than that of starch-internalized proteins and varied depending upon the pretreatment of the turions. Two starch associated proteins were identified immunochemically as α-amylase (EC 3.2.1.1) and the R1 protein (Lorberth et al. (1998) Nature Biotechnology 16: 473–477). Dark-pretreatment of non-dormant turions does not induce starch net degradation. Under these conditions, α-amylase and R1 were bound to the surface of the starch granules. Continuous illumination with red light induces a rapid degradation of starch. Within the first 24 h of illumination the level of starch-associated α-amylase transiently increased and subsequently decreased rapidly. Similarly, the amount of the starch-associated R1 also decreased during illumination. The dissociation of both α-amylase and R1 from the starch granules preceded the decrease in starch content. However, binding of the two proteins to starch granules remained unchanged when the turions did not perform net starch degradation (as observed during continuous darkness, orthophosphate deficiency, or dormancy of the turions). Thus, during net starch degradation, so far unidentified changes are postulated to occur at the surface of the starch particles that are relevant for protein binding. This conclusion was supported by in vitro studies in which the binding of purified β-amylase (EC 3.2.1.2) to starch granules isolated from turions following various pretreatments was monitored. The enzyme did bind to starch granules prepared from dark-stored turions (in which starch degradation had not been initiated), but not to those isolated from illuminated (starch degrading) turions.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 66 (1986), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: α-1,4-Glucan phosphorylase (EC 2.4.1.1) forms from light or dark grown shoots of Pisum sativum L. cv. ‘Kleine Rheinländerin’ have been studied using various electrophoretic techniques. The phosphorylase patterns of green and etiolated shoots differed. Etiolated shoots contained two enzyme forms, one residing inside and the other outside the etioplast; this was shown by electrophoresis of extracts of isolated etioplasts. Purity and intactness of the organelle preparation were ascertained by electron microscopy. Light-grown shoots contained, in addition to these two enzyme forms, a third phosphorylase which appears to be chloroplast-specific. The two plastidic phosphorylase forms differed slightly in their apparent molecular masses (as determined by non-denaturing polyacrylamide gel electrophoresis) and in their affinities towards branched polyglucans (as revealed by affinity electrophoresis). The apparent affinity of the extrachloroplastic phosphorylase form to these polyglucans was orders of magnitude higher than that of the two plastidic enzyme forms. The development of the chloroplast-specific phosphorylase pattern is under photocontrol. Investigations performed with red or far-red illuminated wild-type plants and with a pale mutant which has a highly reduced pigment and thylakoid content suggest that this photocontrol is mediated by phytochrome.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 51 (1981), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In extracts of the unicellular green alga Eremosphaera viridis DeBary (Chlorococcales, Chlorophyceae) the average specific activity of α-1,4-glucan phosphorylase (E.C. 2.4.1.1) was 200 nmol glucose 1-phosphate formed per min and mg protein. Using continuous and discontinuous electrophoresis on polyacrylamide gels, three phosphorylase forms were found. When the log of the relative mobility of the three enzyme forms was plotted versus the acrylamide gel concentration (Ferguson plot) parallel lines were obtained, indicating that the three enzymes were indiscernible with respect to molecular weight. Electrophoresis on density gradient gels resulted in three activity zones lying close to each other. The relative molecular mass (Mr) of the three enzymes was estimated to be around 180,000 with a difference of less than 7,000 between the small and the large forms.
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  • 5
    ISSN: 1432-2048
    Keywords: Key words:Cucurbita (phloem proteins) ; Phloem protein ; Protein modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS; the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539–1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ.
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  • 6
    ISSN: 1432-2048
    Keywords: Chlorella ; Chloroplast ; Light regulation ; Nucleus ; RNA synthesis ; Transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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: 0.5–5×10−10 mol photons cm−2 s−1) 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 of chloroplastic 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)-1,1-dimethyl-urea, 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.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Planta 145 (1979), S. 69-75 
    ISSN: 1432-2048
    Keywords: Pisum ; Spinacia ; Starch (metabolism, phosphorylases)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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 min per mg protein was found, whereas a lower value (6 nmol per min 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 min 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 min per mg protein) and contained two of these three phosphorylase forms.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Planta 158 (1983), S. 428-436 
    ISSN: 1432-2048
    Keywords: Amylase ; Phosphorylase ; Photosynthesis (transitory starch) ; Spinacia (starch degradation) ; Starch metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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 α-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 α-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.
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  • 9
    ISSN: 1432-2048
    Keywords: Affinity chromatography ; α-Glucan phosphorylase ; Spinacia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The non-chloroplastic α-glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.
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  • 10
    ISSN: 1432-072X
    Keywords: Fructose-1,6-bisphosphatase ; Synechococcus leopoliensis ; Anacystis nidulans ; Isoelectric focusing ; Polyacrylamide gel electrophoresis ; Chromatofocusing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract d-Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity in crude extracts of the blue-green alga Synechococcus leopoliensis (Anacystis nidulans) has been investigated using high resolving electrophoretic and chromatographic separation techniques. Two catalytically active enzyme forms which exhibited isoelectric points of 4.7–4.8 (designated from A) and 4.5–4.6 (designated form B) were resolved by isoelectric focusing. Both enzyme forms acted specifically on fluctose-1,6-bisphosphate. No interconversion between the A and B forms of fructose bisphosphatase activity was detected after refocusing. The apparent molecular weight of the two enzyme forms was determined by non denaturing polyacrylamide gradient electrophoresis; the values were 67,000–70,000 and 60,000–65,000 for A and B, respectively. Both enzyme forms were separated by preparative scale chromatofocusing. Kinetic measurements performed with the separated and partially purified fructose bisphosphatase forms indicated that both enzyme forms differ in their AMP sensitivity. The two enzymes were completely inactivated by the addition of cysteamine and reactivated by dithiols but the reactivation kinetics were different.
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