Shankar V, Naziruddin B, Reyes de la Rocha S, Sachdev G P
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Biochemistry. 1990 Jun 19;29(24):5856-64. doi: 10.1021/bi00476a030.
Hydrophobic binding properties of purified human respiratory mucins were studied by the fluorescence probe technique using mansylphenylalanine (Mns-Phe) as the fluorescent probe. Mucins were purified from tracheobronchial secretions of cystic fibrosis (CF) and asthmatic patients, as well as from individuals with normal lungs, according to a protocol earlier established in our laboratory. Purified mucins were subjected to reduction-alkylation and Pronase digestion to study the effects of these treatments on the hydrophobic properties of the mucins. In addition, the effects of increased NaCl concentration on the hydrophobic properties of native and reduced-alkylated mucins were also investigated. Native mucins showed evidence of a large number of low-affinity (KD approximately 10(-5) M) binding sites for the hydrophobic ligand Mns-Phe and had between 40 and 50 binding sites/mg of mucin. Reduction of mucin using dithiothreitol in the presence of 6 M guanidine hydrochloride and subsequent alkylation with iodoacetamide apparently caused marked conformational changes in the mucin molecules as revealed by the presence of both high-affinity (KD approximately 10(-6) M) and low-affinity (KD approximately 10(-5) M) binding sites for the probe and an increase in the number of probe binding sites. Pronase digestion of the native and reduced-alkylated mucins almost completely eliminated binding of the fluorescent probe to the mucins, showing that the binding sites are on the nonglycosylated, Pronase-sensitive portion of the mucin molecules. Increasing NaCl concentrations (0.03-1.0 M) did not appreciably alter the native mucin-induced Mns-Phe fluorescence, while that of the reduced-alkylated mucin-induced Mns-Phe fluorescence was progressively increased.(ABSTRACT TRUNCATED AT 250 WORDS)
采用荧光探针技术，以甲基磺酰苯丙氨酸（Mns-Phe）作为荧光探针，研究了纯化的人呼吸道黏蛋白的疏水结合特性。按照我们实验室先前建立的方案，从囊性纤维化（CF）患者、哮喘患者以及肺部正常个体的气管支气管分泌物中纯化黏蛋白。对纯化的黏蛋白进行还原烷基化和链霉蛋白酶消化处理，以研究这些处理对黏蛋白疏水特性的影响。此外，还研究了增加氯化钠浓度对天然和还原烷基化黏蛋白疏水特性的影响。天然黏蛋白显示出大量对疏水配体Mns-Phe具有低亲和力（KD约为10^(-5) M）的结合位点，每毫克黏蛋白有40至50个结合位点。在6 M盐酸胍存在下用二硫苏糖醇还原黏蛋白，随后用碘乙酰胺进行烷基化处理，结果表明黏蛋白分子发生了明显的构象变化，表现为对探针同时存在高亲和力（KD约为10^(-6) M）和低亲和力（KD约为10^(-5) M）的结合位点，且探针结合位点数量增加。对天然和还原烷基化黏蛋白进行链霉蛋白酶消化，几乎完全消除了荧光探针与黏蛋白的结合，表明结合位点位于黏蛋白分子的非糖基化、对链霉蛋白酶敏感的部分。增加氯化钠浓度（0.03 - 1.0 M）对天然黏蛋白诱导的Mns-Phe荧光没有明显影响，而还原烷基化黏蛋白诱导的Mns-Phe荧光则逐渐增加。（摘要截短于250字）
