Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Abstract

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K.) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the Vm.x is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (Ki) of 2.48 mm.Seeds of faba bean (Vicia faba L.) store approximately 35%

Sucrose synthase expression pattern in the rhythmically growing shoot of common oak (Quercus robur L.)

“…In order to reduce the aspecific linkage, slides were incubated for 20 min with an unconjugated anti-rabbit antiserum raised in goat diluted 1/50 in TBS (Sigma) before incubation for 2 h at room temperature with the primary antibody (anti-rabbit polyclonal antibody given by Ross et al [22]) or with the pre-immune serum diluted 1/500 in blocking solution. The slides were then washed six times in TBS and incubated for 20 min in unconjugated anti-rabbit antiserum in goat diluted 1/50 in TBS before incubation for 1 h at room temperature with the secondary antibody (Goat anti-rabbit IgG alkaline phosphatase conjugate, Promega, France) diluted 1/7500 in TBS.…”

“…The immunological detection of SuSy was performed using a polyclonal antibody raised against the denatured SuSy protein of Vicia faba (a gift from Dr Ross H.A. [22]) diluted at 1/1000 prior to incubation with anti-rabbit IgG (Sigma). The protein-antibody complex was detected using chemiluminescence ECL, western-blotting system (Bio-rad).…”

Sucrolytic Enzyme Activities in Cotyledons of the Faba Bean (Developmental Changes and Purification of Alkaline Invertase)

“…Two booster injections, with the addition of an equal volume of incomplete Freund's adjuvant, were given 27 and 56 d later. Serum was collected 11 d after the final injection and partially purified as described previously (Ross and Davies, 1992a). …”

“…Western blots (using the polyclonal antibodies raised against the denatured alkaline invertase protein) were carried out as described previously (Ross and Davies, 1992a) but with dilutions of antibodies at 1:500 and 1:lOOO. The specificity of the antibodies was initially checked on a western blot with SDS-denatured crude total NaCl protein extract and with preimmune serum as a control.…”

“…The purification and characterization of SUC synthase from bean cotyledons has been reported previously (Ross and Davies, 1992a), but the potential and relative roles of Suc synthase and invertases in contributing to Suc metabolism in developing bean seed have not been addressed adequately. The importance of SUC synthase as a determinant of sink strength has been demonstrated with maize mutants (Chourey and Nelson, 1976) and more recently with transgenic potato tubers, in which Suc synthase is down-regulated and starch biosynthesis is mhibited (Zrenner et al, 1995).…”