Taft C S, Zugel M, Selitrennikoff C P
Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver 80262.
J Enzyme Inhib. 1991;5(1):41-9. doi: 10.3109/14756369109069059.
Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.
用含有5 mM乙二胺四乙酸（EDTA）、1 mM苯甲基磺酰氟、200 mM无机磷酸盐、10 microM鸟苷三磷酸（GTP）、1 mM二硫苏糖醇（DTT）、10 mM氟化钠和600 mM甘油的25 mM 4-（2-羟乙基）-1-哌嗪乙烷磺酸（HEPES）缓冲液（pH 7.4）中的Protargol（0.1% 3-[(3-胆酰胺丙基)-二甲基铵]-1-丙烷磺酸盐）和0.5%辛基葡糖苷处理粗糙脉孢菌原生质体裂解物，分离出葡聚糖合酶活性。通过蔗糖梯度密度沉降对所得活性进行部分纯化。蔗糖梯度峰馏分中约70%的酶活性是可溶的，酶活性纯化了7.3倍。部分纯化的酶活性在4℃下具有数周的半衰期，表观米氏常数（Km(app)）为1.66±0.28 mM。针对粗颗粒和去污剂处理的酶馏分测试了1,3-β-D-葡聚糖合酶活性的抑制剂（西洛芬净、丘疹青霉素B、刺囊霉素A、棘白菌素B、山梨糖和尿苷二磷酸（UDP）），并测定了每种抑制剂的表观抑制常数（Ki(app)）。这种稳定的葡聚糖合酶活性制剂似乎可用于新葡聚糖合酶抑制剂的体外酶筛选。
