Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto |
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Authors: | Adrian Turner Antony Bacic Philip J Harris Steve M Read |
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Institution: | (1) Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia, AU;(2) School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand, NZ;(3) School of Forestry and Resource Conservation, University of Melbourne, Creswick, Victoria 3363, Australia, AU |
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Abstract: | The callose synthase (UDP-glucose: 1,3-β-d-glucan 3-β-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.
Received: 24 September 1997 / Accepted: 12 November 1997 |
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Keywords: | : Callose Callose synthase Nicotiana Plasma membrane Pollen tube Product entrapment |
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