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

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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