Adp glucose

Discontinuous native PAGE was performed as described elsewhere [44] (link). Two different incubation solutions (a and b) were used to visualize starch synthase activities. The incubation buffer (a) contained 50 mM Tricine/KOH (pH 8.0), 25 mM K-acetate, 5 mM DTE, 2 mM EDTA, 0.025% (w/v) bovine serum albumin, and 1 mM ADPglucose (either purchased from Sigma, Taufkirchen, Germany or synthesised using recombinant AGPase from E. coli as described in Fettke et al. [44] (link)). Buffer (a) was used for gels that contained 0.02% (w/v) glycogen from oyster (type II, Sigma). Gels without any glucan were incubated with buffer (b) that contained in addition 500 mM Na-citrate (pH 8.0). After electrophoresis the gels were equilibrated for 20 min in the respective buffer, but ADPglucose was omitted. Gels were incubated overnight at room temperature and stained with iodine.

SS activity was determined essentially as previously described with a protocol [26 (link)] using ADP-glucose instead of UDP-galactose as a glucose donor. Enzyme assays were performed in a 100 μL volume containing 50 mM Bicine, pH 8.5, 25 mM potassium acetate, 2 mM MgCl₂, 0.1% (w/v) bovine serum albumin (BSA), 0.375 mM NADH (Sigma, N8129), 0.7 mM phosphoenolpyruvate tricyclohexylammonium salt (Sigma, P7252), 6 U/ml pyruvate kinase, and 30 U/ml lactate dehydrogenase (both from Sigma, P0294) at 37°C. 10 mM maltotriose (Sigma, M8378) were used as an acceptor. AtSS1 (90–200 nM) was added to a mixture containing the components mentioned above, incubated in a microplate at 37°C for 5 minutes with continuous mixing in the microplate reader and the reaction was initiated by addition of 1 mM ADP-glucose (Sigma, A0627 or enzymatically synthesized as described [30 (link)]). Enzymatic activity was monitored for 1 h as a decrease in NADH absorbance at 340 nm and the linear part of the reaction coordinate was used for activity calculation. Acceptor free or enzyme free samples were used as a blank.