Characterization and partial purification of β-1,3-d-glucan (callose) synthase from barley (Hordeum vulgare) leaves

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The plasma membrane bound β-1,3-d-glucan (callose) synthase, assumed to be involved in the resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei), was partially purified from a microsomal fraction of green barley leaves (Hordeum vulgare L.). Plasma membranes were enriched by aqueous polymer two-phase partitioning of the microsomal fraction in a polyethylene glycol 3350/Dextran T-500 system. The plasma membrane bound callose synthase was dependent on uridine 5′diphosphate (UDP)-glucose (Km 0.39 mM) and was activated by Ca2+, digitonin, cellobiose and polyamines. The enzyme was inhibited by UDP and uridine 5′triphosphate (UTP). Glucanase digestion of the in vitro product showed that it was a β-1,3-linked polysaccharide. Two different procedures were used for further enrichment of polypeptides involved in callose synthase activity. Sucrose gradient centrifugation with concomitant product entrapment showed enrichment of four polypeptides with relative molecular masses (Mrs) of 36, 52, 66 and 170 kDa. Non-denaturing polyacrylamide gel electrophoresis (PAGE) separated the callose synthase from most of the other plasma membrane proteins. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins in the callose activity stained zone revealed six dominant polypeptides with Mrs of 36, 52, 54, 60, 70 an d94 kDa. The 36 and 52 kDa polypeptides were found by both methods suggesting that they could constitute true components of the barley leaf callose synthase. These results are in accordance with previous attempts to isolate the polypeptides involved in callose synthesis from dicot plants.

Original languageEnglish
JournalPlant Science
Volume91
Issue number2
Pages (from-to)127-138
Number of pages12
ISSN0168-9452
DOIs
Publication statusPublished - 1 Jan 1993

    Research areas

  • callose synthase, digitonin, Hordeum vulgare, native gel electrophoresis, plasma membrane, product entrapment

ID: 204470865