In our previous study,a gene predicted to encode a Tyrosine aminotransferase( TAT) was found to be significantly up-regulated in root rot diseased Gerbera by transcriptome sequencing. To confirm the genes and investig...In our previous study,a gene predicted to encode a Tyrosine aminotransferase( TAT) was found to be significantly up-regulated in root rot diseased Gerbera by transcriptome sequencing. To confirm the genes and investigate the function,we cloned the gene by RT-PCR and then conduct bioinformatic analyses. In this study,a 1 537 bp long c DNA sequence of this gene( named as Gh TAT) was firstly cloned,which contained a coding region of 1 233 bp,which was predicted to encode a protein of 410 amino acids. Bioinformatic analysis showed that the Gh TAT was a stable hydrophobic protein without signal peptide. Subcellular location prediction result indicated that this protein located in chloroplast,which is the biosynthesis position of tyrosine and the derived products of tyrosine biosynthesis pathway. Moreover,typical Tyrosine aminotransferase domain was found in this protein,indicating that it is a TAT. According to the TAT-based phylogenetic analysis and similarity analysis,the closest relationship and highest similarity was found between Gh TAT and Halianthus annuus TAT,which again verified the TAT property of Gh TAT. Tyrosine aminotransferase( TAT) is the first enzyme in tyrosine biosynthesis pathway,whose products include many antioxidant substances such as tocopherols and tocotrienols. The up-regulation of Gh TAT in root rot diseased gerbera suggests that it may play an important role in response to the root rot pathogen infection. In addition,60 phosphorylation sites( accounting for 14. 6%) were found in this protein,suggesting that the expression of this protein and its encoding gene were greatly influenced by the phosphorylation reactions.展开更多
Chinese traditional medicine Danshen is the radix of the perennial herbs of Salvia miltiorrhiza Bunge, which has a variety of pharmacological effects and is traditionally and extensively applied clinically to treat ca...Chinese traditional medicine Danshen is the radix of the perennial herbs of Salvia miltiorrhiza Bunge, which has a variety of pharmacological effects and is traditionally and extensively applied clinically to treat cardiovascular disorders. In this research, the genomic genes for tyrosine aminotransferase (TAT) of 38 cultivated populations of Danshen in China were cloned and bioinformatic analyses were conducted to reveal its genetic diversity and phylogeny. The full-length SmTAT was 2296 - 2444 bp including 6 exons (encoding 411 amino acids) and 5 introns. Overall, the SmTAT genes in cultivated Danshen populations are highly conserved with a relative low level of genetic diversity. The spliced exons (1236 bp) had 23 SNP variations with a rate of 1.86%, of which 22 occurred in the white flower S. miltiorrhiza Bge.f.alba population (W-SCHY-W-1) and led to 5 amino acid variations. The entire 290 SNP variations with a rate of 24% in the 5 introns occurred exclusively in W-SCHY-W-1. Phylogenetic trees based on the full-length, combined introns, the spliced exons, and the deduced amino acid sequences of SmTAT all showed a two-clade basic structure with W-SCHY-W-1 uniquely standing alone. The SmTAT gene of the white flower population (W-SCHY-W-1) is unique and especially rich in variations. The first time clarified genomic SmTAT gene structure and genetic diversity in cultivated Danshen populations laid an excellent foundation for further studies on the biosynthesis of bioactives and the molecular breeding of Danshen as well as in plant tyrosine metabolism.展开更多
Dexamethasone (Dex), a ligand for transcriptional enhancement of tyrosine aminotransferase (TAT) and tryptophan 2,3-dioxygenase (TO) genes, (100 nM) maximally increased these mRNA levels at 12 h and 7 h in primary cul...Dexamethasone (Dex), a ligand for transcriptional enhancement of tyrosine aminotransferase (TAT) and tryptophan 2,3-dioxygenase (TO) genes, (100 nM) maximally increased these mRNA levels at 12 h and 7 h in primary cultured rat hepatocytes and the nuclear fraction, respectively. Lactacystin (5 μM) and epoxomicin (0.5 μM), 26S proteasome inhibitors, significantly suppressed the Dex-dependent maximum increase of TAT and TO mRNA levels in the cells and the nuclear fraction. Electrophoretic mobility shift assay demonstrated that lactacystin did not affect binding of glucocorticoid receptor to glucocorticoid responsive element. Furthermore, lactacystin did not affect the activation of GRE luciferase reporter by Dex transfected to the cells. The results demonstrate that 26S proteasome is positively involved in the Dex-dependent increase of TAT and TO mRNA levels in the cells and suggest that the mechanism of action of 26S proteasome may be degradation of some RNase(s), which breaks down TAT and TO mRNAs.展开更多
Tyrosine aminotransferase(TAT)catalyzes the transamination of tyrosine and other aromatic amino acids.The enzyme is thought to play a role in tyrosinemia type II,hepatitis and hepatic carcinoma recovery.The objective ...Tyrosine aminotransferase(TAT)catalyzes the transamination of tyrosine and other aromatic amino acids.The enzyme is thought to play a role in tyrosinemia type II,hepatitis and hepatic carcinoma recovery.The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases.Mouse TAT(mTAT)was cloned from a mouse cDNA library,and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques.The recombinant mTAT is able to catalyze the transamination of tyrosine usingα-ketoglutaric acid as an amino group acceptor at neutral pH.The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxyphenylpyruvate,phenylpyruvate and alpha-ketocaproic acid as amino group acceptors.Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9Åresolution.The crystal structure revealed the interaction between the pyridoxal-5′-phosphate cofactor and the enzyme,as well as the formation of a disulphide bond.The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent.Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity.The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.展开更多
In this report we studied the relationship between hydrocortisone(F) concentration and its inductive effect on tyrosine aminotransferase (TAT) activity in primary cultures of rat hepatocytes, and the regulation of rat...In this report we studied the relationship between hydrocortisone(F) concentration and its inductive effect on tyrosine aminotransferase (TAT) activity in primary cultures of rat hepatocytes, and the regulation of rat liver high- and low-affinity glucocorticoid receptors (GRH and GRL) by glucocorticoids (GC). When F concentration was 1-10 nmol/L, the dose-response relationship showed a tendency of saturation. TAT activity was much higher when F concentration was between 10 nmol/L and 10 μmol/l. When F concentration reached 10 μmol/L the induction of TAT could be completely inhibited by RU486, a competitive antagonist of GRH and GRL. These results indicate that induction of TAT by high F concentration was mediated by GRL. The concentration of GC in plasma remained over 10-6 mol/L for 3 d by subcutaneous injection of F in polyvinyl alcohol into the rats. The binding capacity (Ro) of GRH decreased significantly 1 h after injection and remained at a low level,while Ro of GRL increased at 1 h, 24 h, 48 h after injection. It may be concluded that GC could down-regulate GRH and up-regulate GRL. The pathophysiological and clinical significance of these results is discussed.展开更多
基金Supported by Science and Technology Plan Major Projects of Fujian Province(2015NZ0002-1)
文摘In our previous study,a gene predicted to encode a Tyrosine aminotransferase( TAT) was found to be significantly up-regulated in root rot diseased Gerbera by transcriptome sequencing. To confirm the genes and investigate the function,we cloned the gene by RT-PCR and then conduct bioinformatic analyses. In this study,a 1 537 bp long c DNA sequence of this gene( named as Gh TAT) was firstly cloned,which contained a coding region of 1 233 bp,which was predicted to encode a protein of 410 amino acids. Bioinformatic analysis showed that the Gh TAT was a stable hydrophobic protein without signal peptide. Subcellular location prediction result indicated that this protein located in chloroplast,which is the biosynthesis position of tyrosine and the derived products of tyrosine biosynthesis pathway. Moreover,typical Tyrosine aminotransferase domain was found in this protein,indicating that it is a TAT. According to the TAT-based phylogenetic analysis and similarity analysis,the closest relationship and highest similarity was found between Gh TAT and Halianthus annuus TAT,which again verified the TAT property of Gh TAT. Tyrosine aminotransferase( TAT) is the first enzyme in tyrosine biosynthesis pathway,whose products include many antioxidant substances such as tocopherols and tocotrienols. The up-regulation of Gh TAT in root rot diseased gerbera suggests that it may play an important role in response to the root rot pathogen infection. In addition,60 phosphorylation sites( accounting for 14. 6%) were found in this protein,suggesting that the expression of this protein and its encoding gene were greatly influenced by the phosphorylation reactions.
文摘Chinese traditional medicine Danshen is the radix of the perennial herbs of Salvia miltiorrhiza Bunge, which has a variety of pharmacological effects and is traditionally and extensively applied clinically to treat cardiovascular disorders. In this research, the genomic genes for tyrosine aminotransferase (TAT) of 38 cultivated populations of Danshen in China were cloned and bioinformatic analyses were conducted to reveal its genetic diversity and phylogeny. The full-length SmTAT was 2296 - 2444 bp including 6 exons (encoding 411 amino acids) and 5 introns. Overall, the SmTAT genes in cultivated Danshen populations are highly conserved with a relative low level of genetic diversity. The spliced exons (1236 bp) had 23 SNP variations with a rate of 1.86%, of which 22 occurred in the white flower S. miltiorrhiza Bge.f.alba population (W-SCHY-W-1) and led to 5 amino acid variations. The entire 290 SNP variations with a rate of 24% in the 5 introns occurred exclusively in W-SCHY-W-1. Phylogenetic trees based on the full-length, combined introns, the spliced exons, and the deduced amino acid sequences of SmTAT all showed a two-clade basic structure with W-SCHY-W-1 uniquely standing alone. The SmTAT gene of the white flower population (W-SCHY-W-1) is unique and especially rich in variations. The first time clarified genomic SmTAT gene structure and genetic diversity in cultivated Danshen populations laid an excellent foundation for further studies on the biosynthesis of bioactives and the molecular breeding of Danshen as well as in plant tyrosine metabolism.
文摘Dexamethasone (Dex), a ligand for transcriptional enhancement of tyrosine aminotransferase (TAT) and tryptophan 2,3-dioxygenase (TO) genes, (100 nM) maximally increased these mRNA levels at 12 h and 7 h in primary cultured rat hepatocytes and the nuclear fraction, respectively. Lactacystin (5 μM) and epoxomicin (0.5 μM), 26S proteasome inhibitors, significantly suppressed the Dex-dependent maximum increase of TAT and TO mRNA levels in the cells and the nuclear fraction. Electrophoretic mobility shift assay demonstrated that lactacystin did not affect binding of glucocorticoid receptor to glucocorticoid responsive element. Furthermore, lactacystin did not affect the activation of GRE luciferase reporter by Dex transfected to the cells. The results demonstrate that 26S proteasome is positively involved in the Dex-dependent increase of TAT and TO mRNA levels in the cells and suggest that the mechanism of action of 26S proteasome may be degradation of some RNase(s), which breaks down TAT and TO mRNAs.
基金supported in part by a research grant from NINDS(NS062836)。
文摘Tyrosine aminotransferase(TAT)catalyzes the transamination of tyrosine and other aromatic amino acids.The enzyme is thought to play a role in tyrosinemia type II,hepatitis and hepatic carcinoma recovery.The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases.Mouse TAT(mTAT)was cloned from a mouse cDNA library,and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques.The recombinant mTAT is able to catalyze the transamination of tyrosine usingα-ketoglutaric acid as an amino group acceptor at neutral pH.The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxyphenylpyruvate,phenylpyruvate and alpha-ketocaproic acid as amino group acceptors.Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9Åresolution.The crystal structure revealed the interaction between the pyridoxal-5′-phosphate cofactor and the enzyme,as well as the formation of a disulphide bond.The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent.Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity.The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.
文摘In this report we studied the relationship between hydrocortisone(F) concentration and its inductive effect on tyrosine aminotransferase (TAT) activity in primary cultures of rat hepatocytes, and the regulation of rat liver high- and low-affinity glucocorticoid receptors (GRH and GRL) by glucocorticoids (GC). When F concentration was 1-10 nmol/L, the dose-response relationship showed a tendency of saturation. TAT activity was much higher when F concentration was between 10 nmol/L and 10 μmol/l. When F concentration reached 10 μmol/L the induction of TAT could be completely inhibited by RU486, a competitive antagonist of GRH and GRL. These results indicate that induction of TAT by high F concentration was mediated by GRL. The concentration of GC in plasma remained over 10-6 mol/L for 3 d by subcutaneous injection of F in polyvinyl alcohol into the rats. The binding capacity (Ro) of GRH decreased significantly 1 h after injection and remained at a low level,while Ro of GRL increased at 1 h, 24 h, 48 h after injection. It may be concluded that GC could down-regulate GRH and up-regulate GRL. The pathophysiological and clinical significance of these results is discussed.
