Induction of tyrosine aminotransferase by Sepharose-insulin

Insulin covalently bound to Sepharose causes a nearly 2-fold increase in tyrosine aminotransferase activity in monolayer cultures of hepatoma cells previously incubated with dexamethasone. The time course of the induction and its resistance to inhibition by actinomycin D is similar to that obtained with free insulin, although approximately 100 times higher concentrations of Sepharose-insulin than free insulin are required to achieve the same stimulation. Control experiments demonstrated that 0.2–2% of the bound insulin is released from the Sepharose during incubation with the cells. Because of the much greater sensitivity of the hepatoma cells to free insulin, however, this is sufficient to account for the majority of the stimulatory effect of Sepharose-insulin on transaminase activity. Our data do not exclude the hypothesis that insulin bound to Sepharose stimulates tyrosine aminotransferase activity in HTC cells, but do indicate the need for caution in the use of insoluble derivatives of insulin to determine whether insulin can exert its effects on specific protein synthesis without entering the cell.     

Chronotypic induction of tyrosine aminotransferase by alpha-methyl-p-tyrosine

αMethyl-p-tyrosine induced hepatic tyrosine aminotransferase activitity to different extents depending upon the time of day of administration of the drug. Maximal induction occurred when α-methyl-p-tyrosine (100 mg/kg) was injected intraperitoneally during the first several hours of the light phase of the daily cycle, but the magnitude of the induction depended on the nutritional state of the animal. Induction was 4 to 5-fold greater in fasting rats. The effect of α-methyl-p-tyrosine on hepatic tyrosine aminotransferase is believed to be mediated by decreases in hypothalamic norepinephrine. This hypothesis was supported by the demonstration that decreasing levels of hypothalamic norepinephrine at times of day when hypothalamic turnover of norepinephrine was greatest resulted in the greatest induction of tyrosine aminotransferase, while lowering hypothalamic norepinephrine at times when turnover was minimal resulted in minimal induction of tyrosine aminotransferase.     

Destabilization of tyrosine aminotransferase by amino acids

Summary Several L-amino acids (tyrosine, glutamate, methionine, tryptophan, and phenylalanine) and penicillamine destabilized purified tyrosine aminotransferase by removing enzyme-bound pyridoxal 5-phosphate. The destabilization was measured as a progressive loss of enzyme activity in samples taken at intervals from a primary mixture that was incubated at 37°C. Each destabilizing amino acid either served as a substrate for this enzyme or was a product of transamination. In contrast, L-cysteine destabilized the enzyme only if liver homogenate was added, which generated polysulfide by desulfuration. Cysteine complexed free pyridoxal-5-phosphate but did not remove it from the enzyme. Other amino acids did not destabilize tyrosine aminotransferase at the concentrations tested.Abbreviations TyrAT tyrosine aminotransferase (E.C. 2.6.1.5) - PLP pyridoxal-5-phosphate     

Stimulation of tyrosine aminotransferase activity by dl-alpha-methyltryptophan.

DL-alpha-Methyltryptophan (alphaMeTrp), a synthetic analogue of tryptophan, has been found to be a potent inducer of hepatic tyrosine aminotransferase activity in the adrenalectomized rat. alphaMeTrp is inactive in vitro. Unlike the action of other known inducers (tryptophan, hydrocortisone, adenosine cyclic 3:5-monophosphate, and glucagon), maximal stimulation of enzyme activity occurs only 16 to 30 hours after alphaMeTrp administration and the activity is still elevated at 96 hours. Only the L isomer of alphaMeTrp is active, and addition of a hydroxyl group to position 5 of the indole ring renders an inactive compound. The induction can be prevented by actinomycin D or cycloheximide but not galactosamine. Administration of alphaMeTrp together with hydrocortisone produced an additive stimulation of enzyme activity. alphaMeTrp given along with glucagon or adenosine cyclic 3:5-monophosphate caused a further but not additive increase in enzyme activity. Tryptophan given along with alphaMeTrp promoted no extra stimulation whatsoever. These data indicate that alphaMeTrp and tryptophan may act via a common pathway which in part requires RNA synthesis. Other enzymes, namely alanine and aspartate aminotransferase, ornithine aminotransferase, ornithine carbamoyltransferase, serine dehydratase, and histidine ammonialyase, were not affected by treatment of rats with alphaMeTrp.     

Induction of tyrosine aminotransferase by blastomogenic metabolites of tryptophan and tyrosine]

A comparitive study was made of the effect produced by endogenous blastomogenic agents (3-oxyanthranylic and paraoxphenyl-lactic acids) and their nonblastomogenic anaogues (anthranylic and phenyl-lactic acids) on the activity of tyrosine-aminotranspherase in the rat liver. Blastomogenic metabolites proved to be capable of inducing sharply the enzyme activity. This phenomenon and data on the role played by the increase in the activity of tyrosine-aminotranspherase and tryptophane-oxygenase in tyrosine and tryptophane catabolism on the way of a possible formation of blastomogenic metabolites permitted to put forward a suggestion on the "chain reaction" of accumulation of the endogenous blastomogenic agents in the organism.     

Hormonal deinduction of tyrosine aminotransferase

The effects of several antagonists of glucocorticoid action on a line of hepatoma cells (HTC strain) have been studied in order to determine their mechanisms of action. The induction of tyrosine aminotransferase by dexamethasone can be partially or totally inhibited if an antagonist is added simultaneously with dexamethasone or some time later. Antagonists, even if they have as much affinity for the cytoplasmic receptor as dexamethasone, must be administered at a 100-fold excess as compared to dexamethasone. Their receptor binding kinetics are not identical to those of inducer steroids: moreover there is no correlation between relative binding affinities and anti-inducing capacities. A short contact between the cells and the antagonist is sufficient to obtain a full antagonistic effect, but the antagonist is inactive if administered and removed from the cells before induction. An interpretation if suggested, considering these results which do not find a satisfactory explanation in the classical theory of receptor action.     

Induction of tyrosine aminotransferase by pyridoxine in Drosophila hydei

Salivary glands incubated in various concentrations of pyridoxine (Vitamin B₆) show increasing tyrosine aminotransferase (TAT) activity at concentrations up to 10^–5 M and then decreasing activity up to 10^–2 M but in all cases the activity is greater than that of the controls. This increase in activity is demonstrable for up to 6 h, the longest period tested, and is dependent on the synthesis of new mRNA. A similar increase in TAT activity is observed in salivary glands subjected to heat shock. Antibodies prepared against purified tyrosine aminotransferase precipitate a peptide of the same molecular weight (40 KD) as that induced by pyridoxine.     

Enhancement of rat brain tyrosine aminotransferase activity by cortisol

Administration of cortisol to rats enhanced brain tyrosine:2-oxoglutarate aminotransferase activity by a mechanism excluding stabilization, but possibly involving enhanced synthesis, of the enzyme.     

Insulin-mediated inhibition of the induction of tyrosine aminotransferase by dexamethasone.

Insulin-mediated regulation of glucocorticoid-induced expression of the liver-specific gene tyrosine aminotransferase was studied in a clone of the Reuber rat hepatoma cells. Insulin inhibited dexamethasone-induced chloramphenicol acetyltransferase expression from approximately 4 kb of TAT 5' flanking sequence. The degree of this inhibition was comparable to the response of the endogenous gene. A construct of approximately 3 kbp of 5' flanking sequence exhibited no significant basal expression but retained sensitivity to glucocorticoids and to insulin inhibition of the glucocorticoid response. Results of further analysis of the insulin response in deletion constructs and constructs containing glucocorticoid responsive elements ligated to a heterologous promoter suggest that in addition to the glucocorticoid response elements a region close to the start site in the TAT promoter is necessary for insulin to inhibit glucocorticoid-mediated induction of expression.     

Stabilization of rat liver tyrosine aminotransferase by tetracycline.

Rat liver tyrosine aminotransferase was purified 200-fold and an antiserum raised against it in rabbits. 2. Hepatic tyrosine aminotransferase activity was increased fourfold by tyrosine, twofold by tetracycline, 2.5-fold by cortisone 21-acetate and ninefold by a combination of tyrosine and cortisol administered intraperitoneally to rats. 3. Radioimmunoassay with 14C-labelled tyrosine aminotransferase, in conjunction with rabbit antiserum against the enzyme, revealed that cortisol stimulates the synthesis of the enzyme de novo, but that tetracycline has no such effect. 4. Incubation of rat liver homogenates with purified tyrosine aminotransferase in vitro leads to a rapid inactivation of the enzyme, which tetracycline partially inhibits. 5. The inactivation is brought about by intact lysosomes, and the addition of 10mM-cysteine increases the rate of enzyme inactivation, which is further markedly increased by 10mM-Mg2+ and 10mM-ATP. Here again tetracycline partially inhibits the decay rate, leading to the inference that the increase of tyrosine aminotransferase activity in vivo by tetracycline is brought about by the latter inhibiting the lysosomal catheptic action.