Hydrophobic erythrocyte folate binding proteins are converted to hydrophilic forms by trypsin in vitro.
Hydrophobic erythrocyte folate binding proteins are converted to hydrophilic forms by trypsin in vitro.
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DOI：
10.1016/0005-2736(89)90523-3
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发表时间：
1989-02
期刊：
影响因子：
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通讯作者：
A. Antony;R. Verma
中科院分区：
文献类型：
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作者：
A. Antony;R. Verma
人红细胞膜含有高亲和性叶酸结合蛋白（FBPs），在Triton X-100中用Sephacryl S-200凝胶过滤时，用去污剂增溶后，FBPs分解为表观160 000 Mr部分。这些FBPs与来自其他人体组织的微粒FBPs具有相同的抗原和配体结合特性。在确定这些FBP在红细胞上的矢量方向的研究期间，我们用250 μg胰蛋白酶/ml包装细胞胰蛋白酶化完整细胞，并定量分析上清液中剩余的细胞相关的FBP以及蛋白水解的FBP的产物。用胰蛋白酶孵育完整细胞导致其结合125 I标记的pteroyglutamate（组胺衍生物）的能力呈剂量依赖性降低;在250 μg/ml胰蛋白酶下，与未用胰蛋白酶处理的对照细胞相比，叶酸结合降低了77%。虽然胰蛋白酶处理的细胞含有明显的160 000 MrFBPs的量比未处理的对照细胞成比例地低，但胰蛋白酶处理的细胞的上清液（可溶相）含有保留叶酸结合能力的Mr = 40 000的单一种类。胰蛋白酶上清液和胰蛋白酶处理的细胞中的FBPs的总和是未处理的细胞中发现的FBPs的87%。溶解的颗粒红细胞FBPs和可溶性FBPs的分析在TritonX-100临界胶束浓度以上的水和2 H2O中，用蔗糖密度梯度超离心法对胰蛋白酶产物进行超离心，结果表明，表观160 000 MrFBPs与去污剂结合（疏水）种类（其在Mr = 40 000下在H2O中沉降），而可溶性FBPs（也在Mr = 40 000下沉降）是亲水的并且不结合Triton X-100。这些是第一个数据表明，疏水性FBPs可以直接转化为亲水性FBPs的胰蛋白酶介导的蛋白水解事件。胰蛋白酶敏感位点可能位于洗涤剂结合位点和含有叶酸结合位点的蛋白质主体（Mr= 40 000）之间的交界处。
Human erythrocyte membranes contain high-affinity folate-binding proteins (FBPs) which on solubilization with detergents resolve into apparent 160 000Mrmoieties on Sephacryl S-200 gel filtration in Triton X-100. These FBPs share antigenic and ligand binding characteristics with particulate FBPs from other human tissues. During studies to define the vectorial orientation of these FBPs on the erythrocytes, we trypsinized intact cells with 250 μg trypsin per ml packed cells and quantitatively analysed the remaining cell-associated FBPs as well as the products of proteolysed FBPs in the supernatant. Incubation of intact cells with trypsin resulted in a dose-dependent decrease in their capacity to bind125I-labelled pteroyglutamate (histamine derivative); at 250 μg/ml trypsin, folate binding was decreased by 77% compared to nontrypsin-treated control cells. While trypsinized cells contained proportionately lower quantities of apparent 160 000MrFBPs than untreated control cells, the supernatant of trypsinized cells (soluble phase) contained a single species ofMr= 40 000 which retained folate binding capacity. The sum of FBPs in trypsin supernatant and trypsin-treated cells was 87% of that found in untreated cells. Analysis of solubilized particulate erythrocyte FBPs and soluble (trypsin product) FBPs by sucrose density gradient ultracentrifugation in H2O and2H2O above the critical micellar concentration of Triton X-100 revealed that apparent 160 000MrFBPs were detergent-binding (hydrophobic) species (which sedimented atMr= 40 000 in H2O) while soluble FBPs (also sedimenting atMr= 40 000) were hydrophilic and did not bind Triton X-100. These are the first data which show that hydrophobic FBPs can be directly converted to hydrophilic FBPs by a trypsin-mediated proteolytic event. The trypsin-sensitive site is likely to be at the junction between the detergent-binding site and the major body of the protein (Mr= 40 000) containing the folate binding site.