Macromolecular properties and polymeric structure of canine tracheal mucins.
Macromolecular properties and polymeric structure of canine tracheal mucins.
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犬气管粘蛋白的大分子特性和聚合结构。
DOI：
10.1042/bj2760525
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Sachdev,GP
中科院分区：
文献类型：
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作者：
Shankar,V;Virmani,AK;Naziruddin,B;Sachdev,GP
从犬气管囊分泌物中高度纯化两种高Mr粘液糖蛋白（粘蛋白）CTM-A和CTM-B，并研究了它们的大分子特性和聚合结构。在 SDS/复合凝胶电泳中，观察到每种粘蛋白的弥散带。使用 6% 凝胶的聚丙烯酰胺凝胶电泳也显示粘蛋白中不存在低 Mr 污染物。化学和氨基酸组成的比较揭示了两种粘蛋白之间的显着差异。使用静态激光光散射技术，发现 CTM-A 和 CTM-B 的重均 Mr 值分别约为 11.0 x 10(6) 和 1.4 x 10(6)。两种粘蛋白在含有 6 M-盐酸胍的缓冲液中均表现出浓度依赖性聚集。在类似的实验条件下，还原烷基化的CTM-A的Mr为5.48 x 10(6)，并且没有表现出浓度依赖性聚集。使用甘露苯丙氨酸作为探针，通过荧光探针技术研究了粘蛋白的疏水性，结果表明存在大量低亲和力（KD 约 10(5) M）结合位点。这些位点似乎位于蛋白质核心的非糖基化区域，因为粘蛋白的链霉蛋白酶消化几乎完全消除了探针结合。 CTM-A 和 CTM-B 的二硫键还原并未显着改变探针结合特性。此外，向缓冲液中添加增加的 NaCl 浓度 (0.03-1.0 M) 仅导致天然粘蛋白和还原烷基化粘蛋白的疏水特性发生微小变化。 CTM-A 被去糖基化，但天然粘蛋白和还原烷基化粘蛋白的疏水特性没有显着变化。使用化学和酶法相结合的方法对 CTM-A 进行去糖基化，没有明显的降解。在 SDS/PAGE 上，估计蛋白质核心的 Mr 约为。 60,000。根据主要粘蛋白 CTM-A 的蛋白质和碳水化合物含量，计算出粘蛋白单体的 Mr 约为 100。 140,000。因此，通过物理方法观察到的高 Mr (11 x 10(6]) 是由于粘蛋白单体亚基的自缔合所致。
Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.