Interphotoreceptor retinoid-binding protein (IRBP) is rapidly cleared from the Xenopus interphotoreceptor matrix.
Interphotoreceptor retinoid-binding protein (IRBP) is rapidly cleared from the Xenopus interphotoreceptor matrix.
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DOI：
10.1006/exer.1998.0633
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发表时间：
1999-04
影响因子：
3.4
通讯作者：
L. Cunningham;L. Yang;F. Gonzalez‐Fernandez
中科院分区：
文献类型：
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作者：
L. Cunningham;L. Yang;F. Gonzalez‐Fernandez
光感受器间基质（IPM）是一种高度组织化的细胞外基质，对视网膜发育和功能至关重要。尽管其成分的浓度受到仔细调节，但人们对这种调节的机制知之甚少。光感受器间视黄醇结合蛋白 (IRBP) 是 IPM 中最丰富的可溶性蛋白成分。尽管其清除率被认为是调节 IPM 内 IRBP 浓度的重要因素，但尚无研究测量其细胞外周转率。在这里，我们确定了非洲爪蟾中基质 IRBP 的周转率。通过测量单次注射放射性标记蛋白质前体标记的蛋白质的放射性损失来估计 IRBP 周转率。为了估计 IRBP 周转率，我们研究了以下问题：(1) 从 IPM 中定量提取 IRBP 用于生化分析。 (2) 放射性标记前体的递送途径以实现体内脉冲标记。 (3)选择标记前体以尽量减少放射性标记的重复利用。使用蛋白质印迹分析、免疫沉淀和免疫电子显微镜，我们发现可以通过简单的盐水洗涤从 IPM 中定量提取 IRBP。通过全身或玻璃体内注射[35S]甲硫氨酸或羧基末端标记的[1-14C]亮氨酸来放射性标记IRBP。基质 IRBP 的比活性通过考马斯蓝染色 SDS-聚丙烯酰胺凝胶的磷光成像或荧光照相法测定。玻璃体内注射示踪剂在实现视网膜放射性标记脉冲方面比全身递送更有效。这可能是由于玻璃体内注射使身体充当放射性标记氨基酸的“水槽”。当通过玻璃体内注射递送放射性标记前体时，使用[35S]甲硫氨酸计算的基质IRBP半衰期为25. 6+/-0.82小时；相比之下，使用[1-14C]亮氨酸为10.7+/-2.9小时。使用[1-14C]亮氨酸的更快的表观IRBP周转是在亮氨酸降解过程中早期脱羧的背景下解释的。我们的结果表明，爪蟾 IPM 体内 IRBP 的快速更新，表明 IPM 是一个不断更新的动态结构。
The interphotoreceptor matrix (IPM) is a highly-organized extracellular matrix critical to retinal development and function. Although the concentrations of its components are carefully regulated, little is known about the mechanisms of this regulation. Interphotoreceptor retinoid-binding protein (IRBP) is the most abundant soluble protein component of the IPM. Although its rate of clearance is thought to be an important factor regulating the concentration of IRBP within the IPM, no study has measured the rate of its extracellular turnover. Here we determine the rate of turnover of matrix IRBP in Xenopus. The rate of IRBP turnover was estimated by measuring the loss of radioactivity from protein labeled by a single injection of a radiolabeled protein precursor. To provide an estimate of the rate of IRBP turnover, we have examined the following issues: (1) Quantitative extraction of IRBP from the IPM for biochemical analysis. (2) Routes of delivery of radiolabeled precursor to achieve a pulse label in vivo. (3) Selection of labeled precursor in order to minimize reutilization of radiolabel. Using Western blot analysis, immunoprecipitation and immuno-electron microscopy, we found that IRBP can be quantitatively extracted from the IPM by a simple saline wash. IRBP was radiolabeled by systemic or intravitreal injection of either [35S]methionine or carboxyl-terminal labeled [1-14C]leucine. The specific activity of matrix IRBP was determined by either phosphorimaging or fluorography of Coomassie blue-stained SDS-polyacrylamide gels. Intravitreal injection of tracer was more effective than systemic delivery in achieving a pulse of radiolabel to the retina. This may be due to intravitreal injection allowing the body to act as a 'sink' for radiolabeled amino acid. When radiolabeled precursor was delivered by intravitreal injection, the calculated half-life of matrix IRBP using [35S]methionine was 25. 6+/-0.82 hr; in contrast, it was 10.7+/-2.9 hr using [1-14C]leucine. The faster apparent IRBP turnover using [1-14C]leucine is interpreted in context of the early decarboxylation of leucine during its degradation. Our results demonstrate rapid turnover of IRBP in the Xenopus IPM in vivo and suggest that the IPM is a dynamic structure undergoing continuous renewal.