CHARACTERIZATION AND PARTIAL-PURIFICATION OF BETA-1,3-D-GLUCAN (CALLOSE) SYNTHASE FROM BARLEY (HORDEUM-VULGARE) LEAVES

The plasma membrane bound beta-1,3-D-glucan (callose) synthase. assume d to be involved in the resistance to the powdery mildew fungus (Erysi phe graminis f.sp. hordei), was partially purified from a microsomal f raction of green barley leaves (Hordeum vulgare L.). Plasma membranes were enriched by aqueous polymer two-phase partitioning of the microso mal fraction.in a polyethylene glycol 3350/Dextran T-500 system. The p lasma membrane bound callose synthase was dependent on uridine 5' diph osphate (UDP)-glucose (K(m) 0.39 mM) and was activated by Ca2+, digito nin, cellobiose and polyamines. The enzyme was inhibited by UDP and ur idine 5' triphosphate (UTP). Glucanase digestion of the in vitro produ ct showed that it was a beta-1,3-linked polysaccharide. Two different procedures were used for further enrichment of polypeptides involved i n callose synthase activity. Sucrose gradient centrifugation with conc omitant product entrapment showed enrichment of four polypeptides with relative molecular masses (M(r)s) of 36, 52, 66 and 170 kDa. Non-dena turing polyacrylamide gel electrophoresis (PAGE) separated the callose synthase from most of the other plasma membrane proteins. Sodium dode cyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the prot eins in the callose activity stained zone revealed six dominant polype ptides with M(r)s of 36, 52, 54, 60. 70 and 94 kDa. The 36 and 52 kDa polypeptides were found by both methods suggesting that they could con stitute true components of the barley leaf callose synthase. These res ults are in accordance with previous attempts to isolate the polypepti des involved in callose synthesis from dicot plants.