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.     

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.     

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.

     

Irreversible inactivation of rat liver tyrosine aminotransferase

Homogenates prepared from rat livers irreversibly inactivate tyrosine aminotransferase, both endogenous and purified exogenous enzyme, in the presence of certain compounds which bind to pyridoxal 5′-P. The rate of inactivation ranged from a half-life of 0.72 to greater than 15 hr. The pyridoxal 5′-P binding compounds may be considered to be structural analogs for α-ketoglutarate or l-tyrosine, both of which are substrates for the enzyme. l-Cysteine and l-DOPA are the most effective compounds tested of each of the two structural analog classes, respectively. Absence of the carboxyl group from l-cysteine or l-DOPA has little effect on the half-life of the enzyme, whereas absence or substitution of the amino group results in an increased enzyme half-life. Absence of the —SH group from l-cysteine or of the 3′-OH group from l-DOPA results in little or no inactivation of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increased to greater than 15 hr). Semicarbazide and hydroxylamine have little effect on the stability of the enzyme. Addition of pyridoxal 5′-P to homogenates incubated with l-cysteine or l-DOPA inhibits the inactivation of the enzyme. However, the addition of cofactor to inactivated enzyme does not restore lost activity.There is a disappearance of antigenic cross-reacting material during inactivation of the enzyme. This loss of specific cross-reacting material occurs at a slower rate than the loss of enzyme activity, indicating that enzymatic activity is lost prior to loss of antigenic recognition. A three-step proposal is presented to explain the data observed in which the first step is a reversible loss of pyridoxal 5′-P from the enzyme, followed by a specific irreversible inactivation of the enzyme, and ending with nonspecific proteolysis or degradation of the inactivated enzyme molecules.     

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 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.     

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.     

Structural and immunochemical properties of rat liver tyrosine aminotransferase

Our results show for the first time sequence data of the N-terminal part of tyrosine aminotransferase. This unblocked form of TATase, which has never been detected before, starts with a serine. This serine was found at position 29 of the primary structure of the enzyme deduced from the cDNA. We suggest that this free N-terminal amino acid is the extremity of a TATase form generated by proteolysis during the process of purification. Thus, proteolysis does not occur at the C-terminal as has been suggested before, but rather at the N-terminal region of the enzyme. To confirm this possibility, a peptide corresponding to the sequence of the seven carboxy-terminal amino acids of TATase was synthesized. It was coupled to ovalbumin or keyhole limpet hemocyanin, and the resulting conjugates were used to raise anti-peptide antibodies. The crude sera obtained were purified and their abilities to recognize TATase in ELISA and dot-blot experiments were proven. Our results demonstrate that the C-terminal part of the enzyme is present and well-recognized by the anti-peptide serum prepared. Furthermore, the anti-peptide serum reacts with TATase without inhibiting its enzymatic activity.