Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane.
Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane.
复制标题
几丁质合成酶在酿酒酵母无细胞匀浆中的定位：壳质体和质膜。
DOI：
10.1073/pnas.85.22.8516
复制
发表时间：
1988
影响因子：
11.1
通讯作者：
Bartnicki-Garcia,S
中科院分区：
文献类型：
--
作者：
Leal-Morales,CA;Bracker,CE;Bartnicki-Garcia,S
我们描述了一种改进的方法分级无细胞提取物的酿酒酵母分离其膜成分的等密度和速度沉降相结合。这些程序用于检查几丁质合成酶（几丁质-UDP乙酰葡糖胺转移酶; EC 2.4.1.16）在来自野生型酵母菌株的指数生长壁细胞的匀浆中的亚细胞分布。几丁质合成酶（Chs 1）的活性主要发现在两个不同的囊泡人口几乎相等的丰度，但具有显着不同的浮力密度和颗粒直径。一个种群含有总几丁质合成酶的45-65%，由于微泡大小（中值直径= 61 nm）和特征性低浮力密度（1.15 g/ml）而被鉴定为壳聚糖体;它也缺乏1，3-β-葡聚糖合成酶活性。第二个群体（35-55%）被鉴定为质膜，因为其高浮力密度（1.22 μ g/ml）、大囊泡尺寸（中值直径= 252 nm）和钒酸盐敏感的ATP酶的存在。该级分与1，3-β-葡聚糖合成酶的主峰共沉淀。还检测到第三种少量几丁质合成酶颗粒。基本上所有的几丁质合成酶在两个囊泡人口是zymogenic的，因此，我们认为这些囊泡作为前体的最终活性形式的几丁质合成酶，其在细胞中的位置尚未明确确定。
We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations. These procedures were used to examine the subcellular distribution of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast. Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters. One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter = 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-beta-glucan synthetase activity. The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase. This fraction cosedimented with the main peak of 1,3-beta-glucan synthetase. A third, minor population of chitin synthetase particles was also detected. Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.