Shankar V, Virmani A K, Naziruddin B, Sachdev G P
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Biochem J. 1991 Jun 1;276 ( Pt 2)(Pt 2):525-32. doi: 10.1042/bj2760525.
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.
从犬气管囊分泌物中高度纯化出两种高分子量黏液糖蛋白（黏蛋白）CTM-A和CTM-B，并对其大分子特性及聚合结构进行了研究。在SDS/复合凝胶电泳中，每种黏蛋白均观察到一条弥散带。使用6%凝胶的聚丙烯酰胺凝胶电泳也显示黏蛋白中不存在低分子量污染物。化学组成和氨基酸组成的比较揭示了这两种黏蛋白之间的显著差异。使用静态激光散射技术，发现CTM-A和CTM-B的重均分子量分别约为11.0×10⁶和1.4×10⁶。在含有6M盐酸胍的缓冲液中，两种黏蛋白均表现出浓度依赖性聚集。在类似实验条件下，还原烷基化的CTM-A的分子量为5.48×10⁶，且未表现出浓度依赖性聚集。以甲磺酰苯丙氨酸为探针，通过荧光探针技术研究黏蛋白的疏水特性，结果显示存在大量低亲和力（KD约为10⁵M）结合位点。这些位点似乎位于蛋白质核心的非糖基化区域，因为用链霉蛋白酶消化黏蛋白几乎完全消除了探针结合。CTM-A和CTM-B二硫键的还原并未显著改变探针结合特性。此外，向缓冲液中添加浓度不断增加的氯化钠（0.03 - 1.0M）只会使天然和还原烷基化黏蛋白的疏水特性发生微小变化。CTM-A通过化学和酶法结合进行去糖基化，且无明显降解。在SDS/PAGE上，估计蛋白质核心的分子量约为60,000。根据主要黏蛋白CTM-A的蛋白质和碳水化合物含量，计算出黏蛋白单体的分子量约为140,000。因此，通过物理方法观察到的高分子量（11×10⁶）是由于黏蛋白单体亚基的自缔合。
