Measurement of intracellular collagen degradation.
Measurement of intracellular collagen degradation.
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细胞内胶原蛋白降解的测量。
DOI：
10.1016/0003-2697(81)90382-1
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发表时间：
1981
影响因子：
2.9
通讯作者：
Carol J. Engels
中科院分区：
文献类型：
--
作者：
Robert S. Bienkowski;Carol J. Engels
通过用[14C]脯氨酸孵育成纤维细胞，并测定低分子量组分中总的[14C]羟基脯氨酸的百分比，来定量新合成的胶原的细胞内降解。有几个问题使这一点变得困难。(1)市售的[14C]脯氨酸经常被[14C]羟脯氨酸污染，必须在使用前进行提纯。(2)盐和[14C]脯氨酸对测定低分子量组分中的[14C]羟脯氨酸有干扰，必须用制备离子交换层析除去。(3)酸解过程中反式到顺式羟基脯氨酸的异构化程度是不同的，必须加以考虑。(4)在加工过程中[~(14)C]羟脯氨酸的损失是不同的；[~H]羟脯氨酸可用作回收的内部测量，即使氚可能在水解过程中丢失。用分析阳离子交换树脂对低分子组分和高相对分子质量组分中的[14C]羟基脯氨酸进行最终定量。用这些方法可以测定新合成的胶原蛋白的降解率，精密度为±3%。
Intracellular degradation of newly synthesized collagen is quantitated by incubating fibroblasts with [14C]proline and determining the percentage of total [14C]hydroxyproline that is present in a low molecular weight fraction. Several problems make this difficult. (1) Commercial [14C]proline is often contaminated with [14C]hydroxyproline and must be purified before use. (2) Salt and [14C]proline interfere with the determination of [14C]hydroxyproline in the low molecular weight fraction and must be removed by preparative ion-exchange chromatography. (3) Epimerization of trans- to cis-hydroxyproline during acid hydrolysis is variable and must be taken into account. (4) Loss of [14C]hydroxyproline during processing varies; [3H]hydroxyproline can be used as an internal measure of recovery, even though tritium may be lost during hydrolysis. An analytic cation-exchange resin is used for the final quantitation of [14C]hydroxyproline in the low and high molecular weight fractions. With these methods, degradation of newly synthesized collagen can be determined with a precision of ± 3%.