Turner A, Bacic A, Harris P J, Read S M
Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria, Australia.
Planta. 1998 Jul;205(3):380-8. doi: 10.1007/s004250050334.
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.
来自烟草（Nicotiana alata Link et Otto）花粉管的胼胝质合酶（UDP-葡萄糖：1,3-β-D-葡聚糖3-β-D-葡糖基转移酶；EC 2.4.1.34）（CalS）负责细胞壁多糖胼胝质的发育调控沉积。对在液体培养中生长的烟草花粉管进行膜制备，并通过密度梯度离心进行分级分离。CalS活性沉淀到梯度的较密区域，约为1.18 g.ml-1，远离高尔基体、内质网和线粒体的标志物，并进入富含ATP酶活性以及在低pH下用磷钨酸染色的膜的级分中。这表明花粉管CalS定位于质膜中。通过向下离心富集的膜中的胼胝质合酶活性用洋地黄皂苷溶解，酶活性增加了3至4倍，然后通过产物截留将溶解的活性进一步富集10倍。完整的程序使最终的CalS比活性比花粉管匀浆的比活性高1000倍。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，在纯化过程中，有几种多肽与CalS活性共分级，一种190 kDa的多肽在产物截留沉淀中富集。
