CHARACTERIZATION OF CELL-WALL PROTEINS FROM YEAST AND MYCELIAL CELLS OF CANDIDA-ALBICANS BY LABELING WITH BIOTIN - COMPARISON WITH OTHER TECHNIQUES
CHARACTERIZATION OF CELL-WALL PROTEINS FROM YEAST AND MYCELIAL CELLS OF CANDIDA-ALBICANS BY LABELING WITH BIOTIN - COMPARISON WITH OTHER TECHNIQUES
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DOI：
10.1128/iai.60.11.4898-4906.1992
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发表时间：
1992-11-01
影响因子：
3.1
通讯作者：
SENTANDREU, R
中科院分区：
文献类型：
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作者：
CASANOVA, M;LOPEZRIBOT, JL;SENTANDREU, R
白色念珠菌ATCC 26555芽生分生孢子和带有芽生分生孢子的芽管通过将细胞与C-14-标记的蛋白水解产物一起孵育进行代谢标记，随后用生物素标记。通过用2-巯基乙醇（betaME）处理和用β-葡聚糖酶（Zypholase）处理后，从两种生长形式的完整细胞中提取双标记（放射性和生物素化）细胞壁蛋白和糖蛋白。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离β ME-和酵母酶-提取物，并在硝酸纤维素纸上进行蛋白质印迹（免疫印迹）。聚丙烯酰胺凝胶用考马斯亮蓝染色并处理用于荧光照相。用过氧化物酶缀合的伴刀豆球蛋白A（ConA）或Extravidin进行Western印迹分析。印迹的蛋白质也与针对来自ATCC 26555菌株的菌丝体细胞壁的甘露糖蛋白组分的多克隆抗体和单克隆抗体反应。用生物素标记允许在非常宽的分子量范围（从650至13 kDa）内鉴定细胞壁蛋白和糖蛋白组分的复杂阵列。这些似乎是真正的细胞壁成分。未被考马斯蓝染色或通过间接ConA-过氧化物酶染色的蛋白质印迹法显示为分离度差的多分散条带的生物素化高分子量糖蛋白在与Extravidin-过氧化物酶结合物反应后被检测为清晰的条带。生物素化的分子保留不变的反应性对刀豆蛋白A，多克隆抗体，单克隆抗体。
Candida albicans ATCC 26555 blastoconidia and blastoconidia bearing germ tubes were metabolically labelled by incubating the cells with C-14-labelled protein hydrolysate and were subsequently tagged with biotin. Double-labelled (radioactive and biotinylated) cell wall proteins and glycoproteins were extracted from intact cells of both growth forms by treatment with 2-mercaptoethanol (betaME) and with beta-glucanases (Zymolyase) after treatment with betaME. The betaME- and Zymolyase-extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotted (immunoblotted) to nitrocellulose paper. Polyacrylamide gels were stained with Coomassie blue and processed for fluorography. Western blot analysis was performed either with peroxidase conjugated-concanavalin A (ConA) or Extravidin. Blotted proteins were also reacted with polyclonal antibodies and monoclonal antibodies against mannoprotein components from mycelial cell walls of the ATCC 26555 strain. Labelling with biotin allowed identification of a complex array of cell wall protein and glycoprotein components within a very wide molecular mass range (from 650 to 13 kDa). These appeared to be genuine cell wall components. Biotinylated high-molecular-mass glycoproteins that were not stained with Coomassie blue or that appeared as poorly resolved polydisperse bands by indirect ConA-peroxidase staining of Western blots were detected as sharply defined bands following reaction with the Extravidin-peroxidase conjugate. Biotinylated molecules retained unaltered reactivities against ConA, polyclonal antibodies, and monoclonal antibodies.