STUDIES ON PLASMA MEMBRANE 1,3-p-D-GLUCAN SYNTHASE OF PEANUT COTYLEDON CELLS

Abstract

l,3-/3-D-glucan synthase (UDP-glucose : 1,3-0-D-glucan 3-/3 -Dglucosyltransferase, EC 2.4.1.34) is a plasma membrane localized enzyme which catalyzes the synthesis of callose (1,3- /3-D-glucan) from UDP-glucose in higher plants. In this study two forms of 1,3-/3-D-glucan synthase (GS-IIA and GS-IIB) have been separated from a plasma membrane fraction from the 7-day old germinating peanut cotyledons using selective solubilization of the enzymes with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose density gradient centrifugation and chromatography on hydroxylapatite column. Both forms were subsequently purified to apparent homogeneity by chromatography on hydroxylapatite (second column) followed by chromatography on DEAE-sephadex A-50. The specific activity of the purified GS-IIA and GS-IIB was increased about 636- and 64-fold, respectively, relative to the crude microsomal membrane fraction. On sodium dodecylsulfate polyacrylamide gel electrophoresis GS-IIA and GS- IIB migrated as a single protein band with molecular masses of 48K and 57K, respectively. Determination of the H2N-terminal amino acid by Edman degradation gave phenylthiohydantoin derivatives of Lleucine for GS-IIA and L-lysine for GS-IIB, indicating the presence of single polypeptides in both enzyme forms. The purified GS-IIA and GS-IIB were found to be quite specific 2 + for UDP-glucose as the glucosyl donor and required Ca , at an optimum concentration of 2-5mM, for activity. The activity of both enzymes was inhibited by nucleotides (ATP, GTP, CTP, UDP and UMP). The enzyme activity was also inhibited by the addition of (i) EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the adition of Ca2+. The reaction product formed during incubation of UDP-[14C] glucose and cellobiose with the purified enzymes was susceptible to digestion by exo-(l,3) -/3-Dglucanase, but was resistant to a- and /3-amylases and to periodate oxidation, indicating that polymer formed was l,3-/3-Dglucan and /3-l,4 and /3-1, 6 linkages were absent. Polyclonal monospecific antibodies against GS-IIA and GS-IIB were raised in rabbits. The immune serum obtained with GS-IIA inhibited the enzyme activity and reacted specifically with GSIIA on immunodiffusion plates. The GS-IIA immune serum also did not cross-react with GS-IIB. These results further confirmed the homogeneity of the GS-IIA preparation. The immune serum made with GS-IIB was found to be completely inactive against both GSIIB and GS-IIA, indicating that the two enzyme forms were immunologically different.