MEMBRANE FRACTIONATION AND ENRICHMENT OF CALLOSE SYNTHASE FROM POLLENTUBES OF NICOTIANA-ALATA LINK ET OTTO

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 Nicotian a alata Link et Otto is responsible for developmentally regulated depo sition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated b y density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g.ml(-1), away fro m markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with p hosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membr anes enriched by downward centrifugation was solubilised with digitoni n, which gave a 3- to 4-fold increase in enzyme activity, and the solu bilised activity was then enriched a further 10-fold by product entrap ment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecy l sulfate-polyacrylamide gel electrophoresis showed that several polyp eptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets.