Figure 1.
SS activities from different ss single knock-out plants.
Protein extracts from wild type plants (Ws, Col) and ss single knock-out lines (AtssI in Ws background, AtssII, AtssIII and AtssIV in Col-0 background) were electrophoretically separated under non-denaturing conditions (55 µg protein per lane). (A) Separation gel containing oyster glycogen was incubated with 1 mM ADPglucose. (B) Glucan free gel was incubated with 1 mM ADPglucose and in presence of citrate. After Incubation overnight at room temperature gel was washed with water and stained with iodine solution. Positions of SSs are indicated. Rubisco protein appears as yellowish band (open triangle).
Figure 2.
SS activities from recombinant SS isoforms.
0.8 µg of rSSs (rSSI, rSSII, rSSIII and rSSIV) were electrophoretically separated under non-denaturing conditions. (A) Separation gel containing oyster glycogen was incubated with 1 mM ADPglucose. (B) Glucan free gel was incubated with 1 mM ADPglucose in presence of citrate. After incubation overnight at room temperature gels were washed with water and stained with iodine solution.
Figure 3.
SS activities from different be knock-out plants.
Leaf protein extracts from Atbe3, Atbe2, Atbe3/2 and additionally from wild type (Ws) and AtssI were electrophoretically separated under non-denaturing conditions (65 µg protein per lane). (A) Glucan free gel was incubated with 1 mM ADPglucose in presence of citrate (buffer b). (B) Separation gel containing oyster glycogen was incubated with 1 mM ADPglucose (buffer a). After Incubation overnight at room temperature gels were washed with water and stained with iodine solution. Open triangle indicates the position of rubisco.
Figure 4.
BE dependent SSI activity and mobility of SSI, BE2 and BE3 protein in native PAGE.
(A–B) Protein extracts from be double knock-out (Atbe3/2) and ss single knock-out (AtssI) lines were mixed prior to electrophoresis using different proportions of protein amounts as indicated (75 µg protein per lane in total). Additionally, protein extracts of the mutants were separately analysed and extracts of wild type (Ws) served as control (65 µg protein per lane). Native PAGE was performed using different separation gel concentrations with either (A) 7.5% [T] or (B) 9.5% [T] acrylamide-bisacrylamide. No glucans were applied to the gel. For SS activity staining, gels were incubated with 1 mM ADPglucose in presence of citrate (buffer b) overnight at room temperature and finally stained with iodine. Open triangle indicates the position of rubisco. (C) Immunodetection of AtSSI, AtBE2 and AtBE3 proteins after re-electrophoresis. Following native PAGE (first dimension) using separation gels with different acrylamide-bisacrylamide concentrations (6.0, 8.0 and 9.5% [T] as indicated) gel stripes were cut and denatured for SDS-PAGE (second dimension). After SDS-PAGE proteins were blotted on nitrocellulose and membranes were cut in three pieces according to molecular weights of AtSSI, AtBE2 and AtBE3. Each of the nitrocellulose pieces were incubated with the respective antibodies (e.g. the nitrocellulose piece with protein marker greater than 95 kDa were incubated with antibody against BE3 whereas the nitrocellulose pieces with protein marker below 72 kDa were incubated with antiSSI). Prestained molecular mass marker served as standard. Black triangles indicate the dye front of the gel stripes from the first dimension. (D) rSSI (0.3 µg) was either mixed in different combinations of rBEs (0.2 µg each) as indicated. Native PAGE and activity staining was performed as described for (A). (E) Proteins (rSSI, rBE2 and rBE3) were mixed in different combinations as indicated (0.8 µg each). After electrophoresis under non-denaturing conditions gel was stained with coomassie.
Figure 5.
20 µg of protein extracts were electrophoretically separated under native conditions using different gel concentrations (A). Gels were incubated with phosphorylase a in presence of glucose-1 phosphate overnight at room temperature and finally stained with iodine. (B) BE activity based on stimulation of phosphorylase a was also tested for recombinant proteins (0.3 µg).
Table 1.
Characterization of endogenous carbohydrates extracted from recombinant enzyme preparations.
Figure 6.
Interaction between rSSI and rBE2 and between rSSI and rBE3.
rSSI/rBE2 (0.07 nmol each) and rSSI/rBE3 (0.35 nmol each) were incubated with 1,500 nmol ADPglucose in a total volume of 50 µl. (A) Reaction was performed without any external applied glucosyl acceptor. Incorporation of glucosyl residues were estimated after amyloglucosidase treatment. Additional to measured incorporation of glucosyl residues, reducing ends were estimated after isoamylase treatment (open bars and right axis). (B) Proteins of rSSI, rBE2 and rBE3 expressed in E. coli ΔglgCAP deletion mutant were incubated without any external glucan applied (closed circles with solid line) or incubated in presence of 25 nmol (glucose equivalents) maltodextrins with range of DP6 to DP30 (maximum peak DP15) (open circle with broken line). (C) rSSI was incubated in presence of maltodextrins (see above) either with native rBE2 or rBE3 (closed circle with solid line) or with denatured proteins (open circle with broken line). Average values and standard deviation from independent experiments with n = 6 (A), n = 4 (B) and n = 3 (C) are given.
Figure 7.
Interaction between rSSI-rSSIV with rBE2 and rBE3.
0.07-rSSIV and rBE2 each or 0.35 nmol of rSSI-rSSIV and rBE3 each were incubated with 1,500 nmol ADPglucose in a total volume of 50 µl without any external applied glucan acceptor. Incorporation of glucosyl residues after different time points was estimated after amyloglucosidase treatment. Average values and standard deviation of four independent experiments are given.
Figure 8.
CLD profiles of glucans formed by the interaction of AtSSI with AtBE2 or AtBE3 in native PAGE.
Protein extracts from Arabidopsis leaves (300 µg per cm lane) of Ws, Col, Atbe3 and Atbe2 were electrophoretically separated using native PAGE. Following electrophoresis gels were incubated in 1 mM ADPglucose in presence of citrate overnight at room temperature. As glucans form a milky precipitate in the gel iodine staining was omitted. Glucan chains below DP6 (DP3–5), representing only 0.8–3.5% of the total peak area, were not reproducible and therefore excluded from further analysis. Average values and standard deviation of four independent experiments are given.
Figure 9.
CLD profiles of both, glucans formed by the interaction of rSSI-rSSIV with rBE2 or rBE3 and of Arabidopsis starches.
(A) Proteins were incubated as described for Fig. 7. At different time points (glucosyl incorporation between 300 and 900 nmol) aliquots were taken and treated with isoamylase. Glucans were labelled with APTS and separated using capillary electrophoresis. Average values and standard deviation of independent experiments with rBE2 interacting with SSs (rSSI n = 8, rSSII n = 6, rSSIII n = 5 and rSSIV n = 6) and rBE3 interacting with SSs (rSSI n = 5, rSSII n = 6, rSSIII n = 5 and rSSIV n = 6) are given. Glucan chains shorter than DP 6 were in parts irregular and below 2% of the total peak area and therefore were excluded from the data. (B) Starch granules from Arabidopsis leaves were extracted from plants harvested at the end of light period. CLD profile of debranched starch from wild type (Ws) and AtssI knock-out line were analysed by HPAEC-PAD. Average values and standard deviation of four independent experiments are given.