Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae.
Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae.
复制标题
几丁质合成酶活性与酿酒酵母原生质体中的壳小体和质膜结合。
DOI：
10.1016/0005-2736(88)90408-7
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
J. Schwencke
中科院分区：
文献类型：
--
作者：
A. Flores Martinez;J. Schwencke
研究了酿酒酵母原生质体匀浆中几丁质合成酶的亚细胞分布。使用温和的破坏方法尽量减少液泡破裂和随后被液泡蛋白酶污染的亚细胞组分。对全纯化或部分纯化的匀浆进行异重蔗糖梯度分离后，发现甲壳素合成酶活性分布在两个不同浮力密度和粒径的不同颗粒组分之间。当使用全匀浆时，装载的几丁质合成酶约52%定位于壳质体微泡群（直径40-110 nm，浮力密度(d) = 1.146 g/cm3）。由于其平均直径大（260 nm），浮力密度高（d= 1.203 g/cm3），并且存在对钒酸盐敏感的atp酶活性，另一个具有26%活性的囊泡群被确定为质膜囊泡。此外，用3h -豆豆蛋白A标记原生质体表面后，标记与假定的质膜囊泡共同沉积。酵母质膜标记对壳质体部分的交叉污染可以忽略不计。在质膜和壳质体中，几丁质合成酶是稳定的，基本上是产酶的。壳质体部分的活化产生长度为100-250纳米的微纤维。我们的研究结果支持这样的观点，即壳质体不是由质膜囊泡产生的，而是在酿酒酵母原生质体中被定义为含有大部分几丁质合成酶的亚细胞器。
The sub-cellular distribution of chitin synthetase was studied in homogenates ofSaccharomyces cerevisiaeprotoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d= 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts ofSaccharomyces cerevisiae.