Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto
| Autor: | Philip J. Harris, Antony Bacic, Steve M. Read, Adrian Turner |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Centrifugation
Plant Science Cell Fractionation chemistry.chemical_compound Tobacco Centrifugation Density Gradient Genetics Gel electrophoresis Nicotiana alata biology Endoplasmic reticulum Cell Membrane Callose Membrane Proteins Sodium Dodecyl Sulfate biology.organism_classification Enzyme assay Plants Toxic Digitonin Solubility chemistry Biochemistry Glucosyltransferases biology.protein Pollen Electrophoresis Polyacrylamide Gel Schizosaccharomyces pombe Proteins Cell fractionation |
| Zdroj: | Planta. 205:380-388 |
| ISSN: | 1432-2048 0032-0935 |
| DOI: | 10.1007/s004250050334 |
| Popis: | The callose synthase (UDP-glucose: 1,3-beta-D-glucan 3-beta-D-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g.ml-1, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets. |
| Databáze: | OpenAIRE |
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