RIBONUCLEASES OF HUMAN-SERUM, URINE, CEREBROSPINAL-FLUID, AND LEUKOCYTES - ACTIVITY STAINING FOLLOWING ELECTROPHORESIS IN SODIUM DODECYL SULFATE-POLYACRYLAMIDE GELS
RIBONUCLEASES OF HUMAN-SERUM, URINE, CEREBROSPINAL-FLUID, AND LEUKOCYTES - ACTIVITY STAINING FOLLOWING ELECTROPHORESIS IN SODIUM DODECYL SULFATE-POLYACRYLAMIDE GELS
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DOI：
10.1021/bi00511a030
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
DEKKER, CA
中科院分区：
文献类型：
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作者：
BLANK, A;DEKKER, CA
人血清、尿、脑脊液和白细胞的RNA酶在RNA铸型十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶中电泳后，通过活性染色进行可视化。通过在100 ℃加热2分钟制备电泳用样品。C在2%SDS和5%巯基乙醇中，将蛋白质解离成其组成多肽链并允许估计MW的条件。发现在磷酸纤维素柱上可分离的血清碱性RNA酶活性的5个峰中的每一个，即，Akagi的RNases 1-5与细胞学上不同的酶相关。这些酶在SDS凝胶中表现出的MW为31，000和28，000（RNA酶1的主要种类）、25，000（RNA酶2）、20，000（RNA酶3）、16，000（RNA酶4）和14，000（RNA酶5）。白细胞的RNA酶活性显示MW为17，000，并显示其R对制备电泳样品（在不含巯基乙醇的2%SDS中）的温度的特征依赖性。在血清中发现与白细胞相似的RNA酶活性，不同于RNA酶1-5。尿RNase活性的异质性低于血清，主要由血清RNase 1和一种类似于白细胞RNase的酶组成。相反，CSF RNA酶活性更复杂，包括与血清RNA酶1-5相似的酶以及在血清中未观察到或在层析后作为次要组分在血清中检测到的其他物质。本文所述的分析方法特别适用于评估RNA酶制剂的异质性和直接比较粗品和纯化样品的RNA酶。
The RNases of human blood serum, urine CSF, and leukocytes were visualized by activity staining after electrophoresis in RNA-cast sodium dodecyl sulfate(SDS)-polyacrylamide gels. Samples were prepared for electrophoresis by heating for 2 min at 100.degree. C in 2% SDS and 5% mercaptoethanol, conditions which dissociate proteins into their constituent polypeptide chains and permit estimation of MW. It was found that each of the 5 peaks of serum alkaline RNase activity separable on phosphocellulose columns, i.e., RNases 1-5 of Akagi is associated with electrophoretically distinct enzymes. The MW exhibited by these enzymes in SDS gels are 31,000 and 28,000 (major species of RNase 1), 25,000 (RNase 2), 20,000 (RNase 3), 16,000 (RNase 4) and 14,000 (RNase 5). The RNase activity of leukocytes displays a MW of 17,000 and exhibits a characteristic dependence of its R on the temperature at which samples (in 2% SDS without mercaptoethanol) are prepared for electrophoresis. An RNase activity like that of leukocytes, distinct from RNases 1-5, is found in serum. Urine RNase activity is less heterogeneous than that of serum, consisting mainly of species like serum RNase 1 and an enzyme similar to leukocyte RNase. Conversely, CSF RNase activity is more complex and includes enzymes resembling serum RNases 1-5 as well as additional species either not observed in serum or detected in serum as minor components following chromatography. The analytical methods described herein are particularly useful for assessment of heterogeneity of RNase preparations and for direct comparison of the RNases of crude and purified samples.