Molecular Genetic Studies on the Enzymes Involved in Amino Acids Metabolism and the Hereditary Diseases Caused by Their Errors

有几种遗传性疾病，它们被定义为氨基酸中间代谢中某些酶的突变导致的错误。本研究的目的是分析编码这些酶的基因的分子结构，以了解这些代谢缺陷的真实数字，以及酶蛋白的功能结构域。在这两年中，取得了以下成果1。组氨酸血症是一种组氨酸脱氨酶紊乱。参与这一反应的组氨酸酶首先是从大鼠肝脏中纯化出来的。然后利用纯化的蛋白质制剂，分离出编码其羧基末端半部分的基因片段，并测定其碱基序列。高碳酸尿症的原因是体质上缺乏同质酸氧化酶。从大鼠肝脏中纯化出该酶，并对其羧基末端编码基因进行了筛选和碱基测序。在口腔性酸尿症中有两种酶：轮转酸磷酸核糖转移酶(PRT)和…更多的奥替替丁5‘-单磷酸(OMP)脱羧酶活性很低。这两种酶在哺乳动物中以双功能蛋白的形式存在，命名为UMP合成酶。从与Ehrlich腹水癌和酵母OMP脱羧酶的氨基酸序列相同的长区中选择了一个寡核苷酸序列(42mer)。以该片段为杂交探针，从小鼠脾中筛选出OMP脱羧酶基因克隆，并最终从人胎盘基因文库中克隆出OMP脱羧酶基因。克隆的人DNA含有人UMP合成酶的完整信息。然后对日本首例遗传性口腔性酸尿症进行了研究。患者细胞中的UMP合成酶mRNA的大小和数量与正常对照细胞中的相同。然而，与对照细胞相比，患者B细胞中两种酶的活性非常低。因此，mRNA水平和酶活性之间的差异可能是由于翻译过程中的错误或产生了结构异常的蛋白质，这增加了易感性和/或活性。为了阐明催化尿酸转化为尿囊素的尿酸酶在动物进化过程中是如何失活的，从基因组DNA文库中克隆了具有活性的大鼠尿酸酶基因，并对其结构进行了分析。该基因全长40kb，由8个外显子组成。较少

There are several inherited diseases which are defined as the errors caused by mutation at certain enzymes in the intermediate metabolism of amino acids. The purpose of this research was to analyze molecular structure of genes coding these enzymes, in order to understand the real figure of these metabolic defects, and the functional domains of enzyme proteins. The following results were obtained during these two years.1. Histidinemia is disorder of histidine deamination. Histidase responsible for this reaction was first purified from rat liver. Then by using the protein preparation purified, a fragment of its cDNA encoding the carboxyl-terminal half was separated, and its base sequence was determined.2. The cause of alcaptonuria is a constitutional lack of homogentisic acid oxidase. The enzyme was purified from rat liver, and cDNA encoding its carboxyl-terminal half was screened and the base was sequenced.3. In orotic aciduria two enzymes, orotate phosphoribosyltransferase (PRTase) and … More orotidine 5'-monophosphate (OMP) decarboxylase have very low activities. These two enzymes are known to exist as bifunctional protein designated as UMP synthase in mammals. An oligonucleotide sequence (42 mer) was chosen from the long region identical for the amino acid sequences of both Ehrlich ascites carcinoma and yeast OMP decarboxylase. Using this fragment as a hybridization probe, OMP decarboxylase cDNA clone was selected from mouse spleen, and finally from human placenta cDNA library. The cloned human DNA was found to contain the whole message for human UMP synthase. Then the first case of hereditary orotic aciduria in Japan was studied. The UMP synthase mRNA from cells of the patient was identical in size and quantity to that from cells of a normal control. However, the activities of two enzymes in the patient's B cells were very low compared to those in control cells. Therefore the difference between the level of mRNA and the enzyme activities may be due to either an error in translation procedures or production of structurally abnormal protein, which has increased liability and/or low act ivities.4. In order to clarify how a uricase, catalyzing the conversion of uric acid to allantoin, was inactivated during animal evolution, the rat uricase gene, which is active, was isolated from genomic DNA library, and its structure was analyzed. It has been found that the gene spans 40 kb and consists of 8 exons. Less