Isolation, identification and characterisation of starch‐interacting proteins by 2‐D affinity electrophoresis

Abstract

A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.

Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb

“…Protein concentration in the supernatant was estimated and 16 μg per sample was loaded onto an 8% non-denaturing polyacrylamide gel. Immunoblotting was carried out as described by Kosar-Hashemi et al (2006). The primary antibodies used were wheat SBE IIa (anti-WBE IIa; Rahman et al , 2001) and wheat SBE IIb (anti-WBE IIb; Regina et al , 2005).…”

Characterization of starch phosphorylases in barley grains

“…Soluble proteins were extracted from barley developing endosperm, germinated grain and mature seed using the buffers and conditions described in Kosar‐Hashemi et al . Zymogram analysis of SP elongating activity was conducted as described by Zeeman et al …”

“…Trypsin protection experiments were performed in a manner similar to that described in Tetlow et al . Proteins were separated on 8% SDS polyacrylamide gels and transferred to nitrocellulose membrane . The blots were probed with the anti‐SPPlas1 polyclonal antibodies and visualized using the ECL TM western blotting detection reagents (Amersham Biosciences) according to the manufacturer's instructions.…”

“…Soluble proteins were extracted from barley developing endosperm, germinated grain and mature seed using the buffers and conditions described in Kosar-Hashemi et al 19 Zymogram analysis of SP elongating activity was conducted as described by Zeeman et al 20 The synthetic peptides SPPlas1 (DSEEAPDAEKEDPE) and SPCyt1 (ESWKGSVRDAKREKGC) that were unique to Pho1 and Pho2 respectively were used to raise polyclonal antibodies.…”

Affinity in Electrophoresis

“…In its lifetime a substantial number of papers and reviews dealing with affinity interactions in electrophoretic techniques have been published in Electrophoresis (Table 1). More than 125 papers 18, 38, 46, 47, 49–53, 69–71, 93, 101, 110, 118, 129–238 with the word affinity in the title and dealing with some aspect of affinity electrophoresis have been published in the journal since 1989, the year it started being indexed in PubMed. Prior to that, i.e.…”