The glycan substrate of the cytosolic (Pho 2) phosphorylase isozyme from Pisum sativum L.: identification, linkage analysis and subcellular localization

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.     

Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme

During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased.     

A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)

The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step ¹⁴C labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- α -arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed.     

Evidence for an association of the early light-inducible protein (ELIP) of pea with photosystem II

The precursor to the nuclear-coded 17 kDa early light-inducible protein (ELIP) of pea has been transported into isolated intact chloroplasts. The location of the mature protein in the thylakoid membranes was investigated after using cleavable crosslinkers such as DSP and SAND in conjunction with immuno-fractionation methods and by application of mild detergent fractionation. We show that ELIP is integrated into the membranes via the unstacked stroma thylakoids. After isolation of protein complexes by solubilization of membranes with Triton X-100 and sucrose density-gradient centrifugation the crosslinked ELIP comigrates with the PS II core complex. Using SAND we identified ELIP as a 41–51 kDa crosslinked product while with DSP four products of 80 kDa, 70 kDa, 50–42 kDa and 23–21 kDa were found. The immunoprecipitation data suggested that the D₁-protein of the PS II complex is one of the ELIP partners in crosslinked products.Abbreviations chl chlorophyll - D₁ herbicide-binding protein - DSP dithiobis-(succinimidylpropionate) - ELIP early light-inducible protein - LHC I and LHC II light-harvesting chlorophyll a/b complex associated with photosystem I or II - PAGE polyacrylamide gel electrophoresis - poly(A)-rich RNA polyadenyd mRNA - PS I and PS II photosystems I and II - SAND sulfosuccinimidyl 2-(m-azido-o-nitro-benzamido)-ethyl-1,3-dithiopropionate - Triton X-100 octylphenoxypolyethoxyethanol     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.     

Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1c1c7.

Both cytosolic and high salt nuclear extracts were isolated from Hepa 1c1c7 cells incubated with 2-azido-3[125I]iodo-7,8-dibromo-dibenzo-p-dioxin ([125I]N3Br2DpD). The [125I]N3Br2DpD-labeled cytosolic fraction was subjected to chemical cross-linking with dimethyl pimelimidate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chemical cross-linking of the cytosolic form of the AhR revealed monomeric (97 kDa), dimeric (185 kDa), trimeric (281 kDa), and tetrameric (327 kDa) complexes. In a time course of exposure to the cross-linking reagent, the largest form given above became the predominant AhR form observed in the cytosolic extracts. The 327 kDa cytosolic species apparently consists of a 97 kDa AhR, an approximately 88 kDa protein, an approximately 96 kDa protein, and an approximately 46 kDa protein. Nuclear extracts from [125I]N3Br2DpD-labeled Hepa 1c1c7 cells were applied to sucrose density gradients. The 6 S nuclear receptor peak fractions were pooled and subjected to chemical cross-linking. Analysis by SDS-PAGE revealed a monomeric (97 kDa) ligand binding protein and a dimeric (182 kDa) complex. This would suggest that the nuclear 6 S AhR consists of a 97 kDa AhR and an approximately 85 kDa protein. These findings would indicate that the AhR exists in cytosol as a tetrameric species, while in the nucleus the AhR exists as a heterodimer.     

The role of backscattering in SHG tissue imaging

We investigate the properties of second-harmonic generation (SHG) tissue imaging for the functional biological unit fascia, skeletal muscle, and tendon. Fascia and Achilles tendon primarily consist of similar collagen type I arrays that can be imaged using SHG microscopy. For muscle, it is the myosin molecules represented within the A bands. For fascia and tendon tissue samples, we observe, in addition to a stronger signal in forward images, vastly different features for the backward versus the forward images. In vivo as well as intact ex vivo thick tissue imaging requires backward detection. The obtained image is a result of the direct backward components plus a certain fraction of the forward components that are redirected (backscattered) toward the objective as they propagate within the tissue block. As the forward and the backward images are significantly different from each other for the imaged collagen type I tissue, it is crucial to determine the fraction of the forward signal that contributes to the overall backward signal. For intact ex vivo SHG imaging of Achilles tendon, we observe a significant contribution of forward features in the resulting image. For fascia, the connective tissue immediately surrounding muscle, we only observe backward features, due to low backscattering in muscle.     

Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism

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.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

ATP- and ATP+ ubiquitin-stimulated proteolysis in rat liver and Yoshida ascites hepatoma.

To gain insight into the activity of cytosolic proteases in tumors, the ATP-dependent proteolysis of cell sap and the ATP- and ubiquitin-dependent proteolysis of Fraction II (a cytosolic subfraction freed of endogenous ubiquitin) were measured in the anaplastic Yoshida ascites hepatoma AH 130. Hepatoma cell sap showed only low, although significant, ATP-stimulated proteolysis, as best seen by comparisons with rat liver made on the basis of wet weight. Much of the basal proteolytic activity of cell sap and of its subfraction enriched in high Mr complexes (Fraction X) peaked near 18S in sucrose gradients. In contrast with cell sap, Fraction II from hepatoma degraded [14C]methylcasein more efficiently than Fraction II from normal liver, but the activities for liver and tumor did not differ on a wet weight basis. Altered polypeptide patterns shown by SDS-PAGE in the Yoshida hepatoma suggested that some abundant hepatoma-specific cytosolic protein might interfere with degradation of the [14C]methylcasein by hepatoma.     

Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization,developmental change,in-vitro translation,and processing

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa).Abbreviations IgG immunoglobulin G - kDa kilodalton - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehüser for help in the immunocytochemical studies.     

Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast

A method for cellular fractionation of Chlamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for cell fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.

Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.

     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts

Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat-stressed plant cells. We have developed an experimental system involving expression of cytosolic class I and class II small heat-stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG-like complexes. Class I and class II small Hsps formed class-specific dodecamers of 210-280 kDa, which, upon heat stress, were incorporated into HSG complexes. Interestingly, class II dodecamers alone could form HSG-like complexes (auto-aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment). By analysing C-terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced auto-aggregation and for recruitment of class I proteins. The class-specific formation of dimers as a prerequisite for oligomerization was analysed by the yeast two-hybrid system. In the presence of the endogenous (tobacco) set of heat-stress-induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Although other, more distantly related, members of the Hsp20 family, i.e. the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo-oligomers of 200-700 kDa, none of them could be recruited to HSG complexes.     

Regulation of hepatic glycogen phosphorylase and glycogen synthase by calcium and diacylglycerol

Incubation of rat hepatocytes with angiotensin II (1 nM) produced a time-dependent accumulation of 1, 2-diacylglycerol and inactivation of glycogen synthase with maximum effects at 10 min. The level of diacylglycerol then gradually declined and the activity of glycogen synthase I returned to control values at 30 min. In contrast, angiotensin II caused an increase in cytosolic Ca2+ and an activation of glycogen phosphorylase which were rapid and transient, reaching maximum values in less than 2 min and then returning to control levels at 15 min. There were excellent correlations between the changes in glycogen synthase I and diacylglycerol levels and between the changes in phosphorylase alpha and cytosolic Ca2+ in these time-course studies. However, there was no correlation between the changes in diacylglycerol and phosphorylase alpha or between the changes in cytosolic Ca2+ and glycogen synthase I. Norepinephrine also caused a slow increase in diacylglycerol and inactivation of glycogen synthase, and a rapid increase in cytosolic free Ca2+ and activation of glycogen phosphorylase. Addition of an alpha1-adrenergic blocker (prazosin or phentolamine) caused rapid decreases in cytosolic free Ca2+ and phosphorylase alpha, but only slowly reversed the inactivation of synthase and accumulation of diacylglycerol. The dose-response curves for norepinephrine and prazosin on glycogen synthase were well correlated with those on diacylglycerol. It is proposed that in liver cells, Ca2+-mobilizing hormones regulate phosphorylase a through a Ca2+-dependent mechanism and inactivate glycogen synthase through the generation of diacylglycerol, at least in part. The data provide additional support for the view that protein kinase C may be important in the regulation of glycogen synthase in liver.     

Multiple forms of endo-1,4-beta-glucanases in the endosperm of Euphorbia heterophylla L

Germinating seeds of Euphorbia heterophylla L. contain endo-1,4-beta-glucanases which degrade carboxymethylcellulose (CMC). The activity decreased approximately 66% in extracts of endosperm containing isopropanol or ethanol. The endoglucanases were isolated from endosperm extracts using ammonium sulphate fractionation followed by Sephacryl S-100-HR chromatography resulting in two main peaks: I and II. Peak I endoglucanase was further purified about 15-fold on DEAE-Sephadex A50 and then by affinity chromatography (CF11-cellulose). Peak II endoglucanases were further purified 10-fold on CM-cellulose chromatography. The results indicated the occurrence of a 66 kDa endoglucanase (fractionated by SDS-PAGE and visualized by activity staining using Congo Red). Several acidic (pI 3.0 to 5.7) and basic (pI 8.5 to 10.0) forms from both peaks which differed in their capacities for degrading CMC or xyloglucans from Copaifera langsdorffii or Hymenaea courbaril were detected.     

Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages of Anisakis simplex

Extracts of Anisakis simplex third (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of alpha-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS-PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: alpha 1, alpha 2 and alpha 3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The alpha-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations of A. simplex larvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.