Ruiz-Herrera J, Sing V O, Van der Woude W J, Bartnicki-Garcia S
Proc Natl Acad Sci U S A. 1975 Jul;72(7):2706-10. doi: 10.1073/pnas.72.7.2706.
Purified preparations of chitin synthetase (EC 2.4.1.16; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase), capable of forming microfibrils in vitro, were isolated from yeast cells of Mucor rouxii. Chitin synthetase was obtained either by substrate-induced liberation of bound enzyme (54,000 x g pellet) or by isolation of unbound enzyme present in the 54,000 x g supernatant of a cell-free extract. Both preparations contained ellipsoidal granules from about 350 to 1000 A diameter. Many granules exhibited a marked depression. No typical unit membrane profiles appeared in thin sections of glutaraldehyde/OsO4-fixed samples. Upon incubation with substrate and activators, chitin microfibrils were produced. The microfibrils were often found intimately associated with granules. The most common configurations were: a microfibril with a granule at one end, or two microfibrils "arising" from the same granule. These findings lend support to the granule hypothesis for the elaboration of cell wall microfibrils by end-synthesis.
从鲁氏毛霉的酵母细胞中分离出了几丁质合成酶（EC 2.4.1.16；UDP-2-乙酰氨基-2-脱氧-D-葡萄糖：几丁质4-β-乙酰氨基脱氧葡萄糖基转移酶）的纯化制剂，该制剂能够在体外形成微纤维。几丁质合成酶可通过底物诱导释放结合酶（54,000×g沉淀）或通过分离无细胞提取物54,000×g上清液中存在的未结合酶来获得。两种制剂都含有直径约350至1000埃的椭圆形颗粒。许多颗粒呈现出明显的凹陷。在戊二醛/四氧化锇固定样品的薄切片中未出现典型的单位膜轮廓。与底物和激活剂一起孵育后，产生了几丁质微纤维。微纤维常常被发现与颗粒紧密相关。最常见的构型是：一端有一个颗粒的微纤维，或从同一个颗粒“长出”的两个微纤维。这些发现支持了通过末端合成来构建细胞壁微纤维的颗粒假说。
