A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

A liver factor that interconverts multiple forms of tyrosine aminotransferase.

Three activity peaks of rat liver soluble tyrosine aminotransferase have been resolved using hydroxyl-apatite chromatography. These peaks interconvert during storage of the soluble enzyme preparation in ice for 20 h. A component of a particulate fraction of liver which will interconvert the forms of tyrosine aminotransferase in vitro with no alteration of total enzyme activity has been detected. This factor is present in a 31, 000 gh pellet of liver and is solubilized by sonication. When the factor is subjected to dialysis or incubation at 25°C for 30 min. its effect on tyrosine aminotransferase is greatly diminished.     

Purification of the native form of tyrosine aminotransferase from rat liver

A procedure that provides a homogeneous, native form of tyrosine aminotransferase (l-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver in exceptionally high yield is described. This goal is accomplished by rapidly inactivating the lysosomal converting factor that generates two additional, lower-molecular-weight forms of tyrosine aminotransferase, and by separating the native enzyme from the altered forms by chromatography on carboxymethyl-Sephadex C-50 and an optional hydroxylapatite step. Homogeneity appears to be achieved after the carboxymethyl-Sephadex C-50 step, and the final yield of the enzyme exceeds 30%.     

Properties of tyrosine aminotransferase from rat liver.

A series of sequential chromatographic procedures which yield essentially homogeneous tyrosine aminotransferase (l-tyrosine:2-oxglutarate aminotransferase, EC 2.6.1.5) from rat livers is described. Analysis of the purified enzyme indicates that its molecular weight is about 100,000, and that it consists of two subunits of identical mass and charge, each bearing one functional site for reaction with pyridoxal phosphate.     

Regulation of tyrosine aminotransferase in foetal rat liver.

A specific tyrosine aminotransferase, separate from the aspartate aminotransferases, is present in low concentration in foetal rat liver at the 21st day of gestation. Intraperitoneal injections of tyrosine methyl ester into the foetuses in utero increase the activity 2-fold, whereas glucose injections decrease it. Tyrosine, dexamethasone and dibutyryl cyclic AMP induce the enzyme activity in organ culture to the same extent as in adult rat liver in vivo.     

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.     

Prenatal induction of tyrosine aminotransferase in rat liver

• 1.1. Hepatic tyrosine aminotransferase activity from adult rat can be resolved into four components on hydroxylapatite column.
• 2.2. A similar profile of enzyme distribution can be obtained from late foetal liver.
• 3.3. Insulin administration to pregnant rats result in induction of two isoenzymes of tyrosine aminotransferase in foetal rat liver. Similarly Cyclic AMP injection to foetal rats in utero results in the induction of the same two forms of the enzyme.
• 4.4. Triaminolone injection to foetal rats in utero leads to the induction of three of the isoenzymes of tyrosine aminotransferase.

     

Diurnal variations in inducibility of rat liver tyrosine aminotransferase activity.

The activity of tyrosine aminotransferase (TAT) was measured in the livers of rats which were entrained to eat for the first 2 hours of a daily 12 hour dark period (‘2+22’ schedule) and were treated with the synthetic glucocorticoid dexamethasone and with glucagon at several times of day. TAT activity in untreated animals varies diurnally with a maximum 4 to 6 hours after the beginning of feeding. In both fed and fasted rats there was a small diurnal variation in inducibility by dexamethasone: in fed rats induction was greatest near the beginning of the dark period, shortly after feeding; in fasted rats induction increased towards the end of the dark period. Glucagon induction showed a marked diurnal variation in fed rats with a decrease coincident with the decline in control TAT activity after its food-induced peak. This variation did not appear to be depemdent on food intake, however, since the decline in inducibility occurred in fasted rats at the same time as in fed rats. Co-treatment with dexamethasone did not affect the decrease in glucagon inducibility. The diurnal variation in TAT induction may reflect a diurnal rhythm in the components of the enzyme synthesizing system (e.g. in the availability of mRNA or in enzyme degradation).     

Stabilization of rat liver tyrosine aminotransferase in vivo by pyridoxine administration.

Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas administration of cortisol, cyclic AMP, glucagon, insulin, tryptophan or tyrosine does not. The results of these and other experiments with pyridoxine are discussed in relation to the mechanisms of action of this vitamin on the activity of the enzyme.     

Diurnal variations in response of rat liver tyrosine aminotransferase activity to food intake

Effects of fasting and refeeding on the hepatic tyrosine aminotransferase activity were examined in rats that had been fed during the night. The tyrosine aminotransferase activity showed clear diurnal variations, with a maximal activity after the feeding time. The tyrosine aminotransferase rhythm persisted even under starvation, though the amplitude decreased remarkably. When the starved rats were refed at night, the tyrosine aminotransferase activity increased rapidly to a high level, but it increased slowly to a rather lower level when they were refed in daytime.     

A simplified procedure for the purification of rat liver tyrosine aminotransferase.

Rat liver tyrosine aminotransferase was purified by chromatography on CM-Sephadex C-50 and DEAE-cellulose, (NH4)2SO4 fractionation and gel filtration on Sephadex G-200. Livers from 400 rats can be easily worked up by this procedure. Furthermore, this purification method has the advantage that hepatic tryptophan 2,3-dioxygenase, which, like tyrosine aminotransferase, is induced by glucocorticosteroids, can be purified from the same homogenate. Tyrosine aminotransferase purified by this method was shown to be specific for 2-oxoglutarate. Its subunits have a molecular weight of 45 000. The following "apparent" Michaelis constants were determined: L-tyrosine, 1.7 X 10(-3) M; 2-oxoglutarate, 5.9 X 10(-4) M; and pyridoxal 5'-phosphate, 2.1 X 10(-6) M. Tyrosine aminotransferase, depleted of its cofactors, binds 4 molecules of pyridoxal 5'-phosphate per 90 000 daltons with a KA of 2.2 X 10(5) M-1.     

L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

Capillary electrophoresis assay of alanine:glyoxylate aminotransferase activity in rat liver

We measured hepatic alanine:glyoxylate aminotransferase (AGT) activity using capillary electrophoresis. After rat liver homogenate was incubated in the presence of substrates and pyridoxal 5'-phosphate, the pyruvate and glycine produced by AGT were measured. The AGT activity was 10.02+/-0.31 micro mol pyruvate/h/mg protein and 10.21+/-0.15 micro mol glycine/h/mg protein. This method is relatively simple and shows superior sensitivity, allowing the measurement of enzyme activity in 5 micro g of protein. Therefore, it appears to be suitable for laboratory use and may also have advantages for measuring AGT activity in liver biopsy specimens.