Hanna R, Brummell D A, Camirand A, Hensel A, Russell E F, Maclachlan G A
Biology Department, McGill University, Montreal, Quebec, Canada.
Arch Biochem Biophys. 1991 Oct;290(1):7-13. doi: 10.1016/0003-9861(91)90584-6.
GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.
来自豌豆（Pisum sativum）上胚轴微粒体膜的GDP-岩藻糖：木葡聚糖1,2-α-L-岩藻糖基转移酶很容易被两性离子去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（Chaps）提取溶解。以GDP-[14C]岩藻糖作为岩藻糖基供体，罗望子木葡聚糖（XG）作为受体时，在0.3%（w/v）的Chaps浓度下观察到最大激活，在0.4%时溶解活性产量最高。反应产物被木霉纤维素酶水解，产生标记的寡糖，这些寡糖在凝胶渗透色谱上的洗脱体积与豌豆XG九糖和十糖亚基相同处达到峰值。膜结合或溶解酶将岩藻糖基转移到罗望子XG的表观Km约为80μM GDP-岩藻糖。这是岩藻糖基转移到内源性豌豆新生XG表观Km的10倍。最佳活性在pH 6至7之间，等电点接近pH 4.8。溶解的酶不需要添加阳离子或磷脂，也不受其刺激，并且在-70℃下可稳定保存数月。相对于微粒体，在Sepharose CL-6B柱上进行溶解和凝胶渗透色谱可使酶的比活性提高约20倍。在CL-6B柱上分级分离的活性，其表观分子量为150 kDa。将溶解的岩藻糖基转移酶在含有0.02%（w/v）罗望子XG的非变性聚丙烯酰胺平板凝胶上进行电泳，通过在GDP-[14C]岩藻糖中孵育、洗涤并对凝胶进行放射自显影来定位其活性。出现一条标记反应产物的条带，表观分子量为150 kDa。
