Dopa decarboxylase (DDC; aromatic-Lamino-acid decarboxylase; aromatic-L-amino-acid carboxylyase, EC 4.1.1.28) was purified from rat liver and its partial sequence was determined. Synthetic oligonucleotides were used to construct and screen rat liver cDNA libraries, and three clones were isolated and sequenced. The 2 kilobases of DDC cDNA cloned consisted of a 5'-noncoding segment of 78 nucleotides, a coding region of 1440 nucleotides, and a 3'-noncoding region of 438 nucleotides. The encoded protein of 480 amino acid residues had a molecular weight of 54,000. A special feature of the primary structure of rat DDC was a repeating structure consisting of 29 amino acid residues. A sequence of 58 amino acid residues, including this repeating structure of rat DDC, was found to show homologies with those of rat tyrosine hydroxylase, human dopamine j3-hydroxylase, and bovine phenylethanolamine N-methyltransferase, other mammalian enzymes that synthesize catecholamines. These results indicate that catecholamine biosynthetic enzymes are structurally related and suggest that their homologous domains are important for catechol-protein interactions.The biosynthetic pathway of catecholamines has been well established and four enzymes-tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], dopa decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; aromatic-L-amino-acid carboxy-lyase, EC 4.1.1.28), dopamine P-hydroxylase [DBH; dopamine f3-monooxygenase; 3,4-dihydroxyphenethylamine, ascorbate: oxygen oxidoreductase (P-hydroxylating), EC 1.14.17.1], and phenylethanolamine N-methyltransferase (PNMT; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28)-are known to be involved in biosynthesis of epinephrine from tyrosine. Recently, three of these enzymes (all but DDC) were cloned (1-3) and it became possible to investigate the regulation of these steps of catecholamine synthesis at the molecular level. DDC catalyzes decarboxylation not only of dopa to dopamine but also of 5-hydroxytryptophan to serotonin (4-7), and it is the sole enzyme necessary in both catecholamine and indoleamine biosynthesis. The enzyme is found in neural tissues and also in peripheral organs, especially liver and kidney. But there are no reports concerning the regulation of DDC expression in neural tissue, nor is it known why high activity of DDC is expressed in liver and kidney. Recently, the DDC gene of Drosophila was cloned and sequenced (8), but in Drosophila >90% ofthe DDC is concerned with cuticle sclerotization (9).We have purified rat liver DDC, obtained polyclonal and monoclonal antibodies to it, and determined some of its enzymological properties (10). We have also studied the difference between rat DDC and rat histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22), which synthesizes histamine (11). Molecular cloning and sequencing of rat DDC cDNA should be useful in understanding the regulation of the mammalian DDC gene,...
cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.
Induction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increase in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4.1.1.32] for gluconeogenesis.
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