Genetic analysis of human deoxyribonuclease I by immunoblotting and the zymogram method following isoelectric focusing.
Genetic analysis of human deoxyribonuclease I by immunoblotting and the zymogram method following isoelectric focusing.
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通过免疫印迹和等电聚焦后的酶谱法对人脱氧核糖核酸酶 I 进行遗传分析。
DOI：
10.1016/0003-2697(89)90175-9
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发表时间：
1989
影响因子：
2.9
通讯作者：
K. Kishi
中科院分区：
文献类型：
--
作者：
T. Yasuda;K. Mizuta;Y. Ikehara;K. Kishi
我们已经设计了两个独立的检测方法，调查可能的分子异质性和遗传多态性的人DNA酶I，在其抗原性和酶活性。一种是免疫印迹法，使用特异性抗体的DNA酶I聚丙烯酰胺凝胶等电聚焦（IEF-PAGE）。使用从人尿中纯化的酶作为免疫原，在兔中产生DNase I特异性抗体。尿中的DNA酶I被发现以多种形式存在，具有不同的pI值，可通过IEF-PAGE在pH 3.5-4.0范围内分离。该方法可检出0.1 μg纯化的DNase Ⅰ，并可根据DNase Ⅰ同工酶谱的不同将不同个体的去唾液酸化尿样分为几组。约0.5ml的原始尿液足以用于同工酶谱的分析。另一种方法是酶谱法，它具有高灵敏度和分辨率几乎相同的免疫印迹法分析DNA酶I模式。它更容易执行，更省时，更有用，因为它不需要对DNA酶I特异性的抗体。这两种方法应证明有价值的生化和遗传分析的DNase I同工酶。
We have devised two independent detection methods for investigating possible molecular heterogeneity and genetic polymorphism in human DNase I, in terms of both its antigenicity and enzymatic activity. One was an immunoblotting method using an antibody specific to DNase I following polyacrylamide gel isoelectric focusing (IEF-PAGE). The DNase I-specific antibody was raised in a rabbit using purified enzyme from human urine as the immunogen. DNase I in urine was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 3.5–4.0. This method was able to detect as little as 0.1 μg of the purified DNase I and facilitated classification of desialy-lated urine samples from different individuals into several groups according to differences in DNase I isozyme patterns. About 0.5 ml of the original urine was sufficient for analysis of the isozyme patterns. The other method was the zymogram method, which had a high sensitivity and resolution almost identical to those of the immunoblotting method for analysis of DNase I patterns. It was easier to perform, more time-saving, and more useful since it did not require antibody specific to DNase I. These two methods should prove valuable for biochemical and genetic analysis of DNase I isozymes.