Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development
Fig 5
De novo production of α-1,4 glucans by HvPho1.
(A) Proton NMR analysis of de novo synthesis of α-1,4 glucans by HvPho1. HvPho1 and HvPho1Asp383Ala (0,1 mg∙ml-1) were incubated with G1P (25 mM) as sole substrate. The figure shows production of 1,4 glycosidic bonds, usage of G1P and generation of reducing ends recorded over time by proton NMR spectroscopy. (B) Plot of the generation of 1,4 glycosidic linkages over the number of reducing ends indicating approximate lengths of glucans produced over time. (C) Recombinant HvPho1 (0.05 mg∙ml-1) and HvBeIIa (0.05 mg∙ml-1) were incubated with G1P (50 mM). Samples were taken at the start of the reaction (0 h) after 2 h and after 4 h. (D) The last sample (4 h) from panel C was debranched using CrIsa1 (0.01 mg∙ml-1) at 25°C overnight. Each sample was boiled, filtered and loaded on a CarboPac PA-100 ion-exchange column and analyzed with pulsed amperometric detection. The gradient used could not distinguish between species with more than 32 glucoses.