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Inorganic phosphate (P(i)) plays a key role in diverse physiological functions. Recent studies have indicated that P(i) affects Akt signaling through the sodium-dependent phosphate cotransporter. Akt signaling, in turn, plays an important role in liver development; however, the effects of high dietary P(i) on the liver have not been investigated. Here, we examined the effects of high dietary phosphate on the liver in developing mice. We found that high dietary P(i) increased liver mass through enhancing Akt-related cap-dependent protein translation, cell cycle progression, and angiogenesis. Thus careful regulation of P(i) consumption may be important in maintaining normal development of the liver.

Microsomal preparations from adult male rat liver actively oxidized RU38486 into the 11 beta-monodemethylated, 11 beta-didemethylated and 17 alpha-hydroxylated derivatives, metabolites which are known to be formed in vivo. These oxidative reactions were inhibited at different degrees by P450 chemical inhibitors. Pretreatment of the animals by P450 mono-oxygenase prototype inducers led to drastic changes in RU38486 metabolization. Methylcholanthrene treatment carried out a significant decrease while phenobarbital markedly increased the metabolic activity of the liver microsomes. Moreover, antibodies to methylcholantrene-inducible P450 forms did not affect the metabolic activity while a complete blockade-of RU38486 oxidation was observed in the presence of antibodies to phenobarbital- inducible forms. The present results demonstrate that liver P450 mono-oxygenases are engaged in different oxidative steps of RU38486 metabolism and that phenobarbital-inducible but not methylcholanthrene-inducible P450 forms are active in RU38486 degradation.

Antisera directed against synthetic peptides with sequences that correspond to selected regions of tyrosine aminotransferase may react with the protein without affecting its biological activity. The antiserum against the theoretical N-terminal dodecapeptide recognizes the enzyme and makes it possible to detect a blocked form of the enzyme. Another form shortened by seven aminoacids and starting with Thr 8 has been found. The isolation of tyrosine aminotransferase by one step affinity chromatography is now made possible; nevertheless the elution procedure remains a critical point. The strategy described should have further applications and allow the detailed exploration of the essential domains of aminotransferases, especially those involved in the function and the degradation of pyridoxal phosphate requiring enzymes.

We have studied tyrosine aminotransferase in the liver of adult and old rats. Thermostability and trypsin action were not modified, and no charge differences have been found by isoelectric focusing between 'adult' and 'old' enzymes. Inducibility by glucocorticoids was increased in vivo in these 27- to 31-month-old rats, but not in vitro, in cultured hepatocytes. Moreover, we have shown the absence of 'cross-reacting material' for tyrosine aminotransferase in senescent rat livers. The rapid turnover of this enzyme may explain the apparent absence of alterations during aging.

In an attempt to clarify the significance of the separable forms of tyrosine aminotransferase, the enzyme from rat liver and from cultured hepatoma cells was studied by carboxymethyl-Sephadex chromatography. Our studies of the form conversion during the purification procedure of the enzyme, where all cellular components were quickly discarded, do not allow us to invoke a specific "converting factor", the existence of which in the particulate fraction has been suggested. Moreover the addition of serine protease inhibitors is not sufficient to prevent the classical conversion. More probably, several factors depending on the environmental conditions might influence different reactions which lead to a preferential conformation of the enzyme in vitro. The difference in the PO4- content of the various enzyme forms and the consecutive differences in negative charge may be the determining factor in the elution pattern of the three forms of the isolated soluble enzyme. This observation raises the possibility that phosphorylation might play a specific role in the regulation of tyrosine aminotransferase synthesis.

The metabolism of the potent antagonist RU38486 has been studied in cultured liver cells and in two hepatoma cell lines. In the liver cells, this steroid undergoes a rapid degradation, whereas in hepatoma cells grown in similar conditions only a minor degradation occurs. Moreover the rate of degradation is much higher for the antagonist steroid than for the agonist steroid tested, dexamethasone. The high antiglucocorticoid potency and the relative instability of the RU38486 molecule are very important to define its different effects and its mechanism of action in liver and in liver derived tumor cells. RU38486 may represent a useful drug in cancerotherapy.

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.

Tyrosine aminotransferase was induced in adult and senescent rat liver and its properties studied. We show the appearance of a 'cross-reacting material' for induced tyrosine aminotransferase of old rats compared to basal enzyme; this cross-reacting material can be provoked in adult rats after injection of cycloheximide, and suppressed in adult and old rats after injection of a serine protease inhibitor (tosylphenylalanine chloromethylketone). Other properties of induced tyrosine aminotransferase (thermostability, Km for tyrosine, isoelectrofocusing) are identical except for the proportion of the three forms and their sensitivity to trypsin in the absence of pyridoxal phosphate, which is increased in senescent animals. The suppression of cross-reacting material clearly indicates that it is not due to errors on old rat liver DNA but rather to post-translational modifications. This demonstrates also the role of serine proteases in tyrosine aminotransferase degradation. We suggest that induced enzyme of senescent rats would undergo a conformational change, possibly due to a release of pyridoxal phosphate from the enzymic molecules, which would thus become more susceptible to proteolytic attack than those of adult rats.

Multiple form patterns of tyrosine aminotransferase were studied in senescent and adult rat liver. Two main modifications were described for "old" rat enzyme: (i) appearance of a new molecular form, specific for old rats, eluted after adult form III and having other properties identical to this form; (ii) disappearance of intermediate forms II and III after enzyme induction; this result seems to be due to acceleration of the conversion process. Vitamin B6 deficiency of old rats explain this and other (previously described) post-translational modifications of "old" tyrosine aminotransferase. The influence of pyridoxal 5' phosphate and the role of protease(s) in the multiple form conversion are discussed. Moreover we show the possibility of a correlation between in vivo alterations of the enzyme molecule and modifications of tyrosine aminotransferase multiple form patterns observed in vitro.

In an attempt to elucidate the relationship between the antiglucocorticoid effect and the state of differentiation of the target cells, we studied the metabolism of the potent antagonist in cultured liver and hepatoma cells (HTC, FAZA). After incubation of [3H]RU38486 with the cells for different periods of time, the native steroid and its metabolites were extracted and analyzed by thin layer chromatography. We observed that RU38486 was not metabolized in the transformed cell lines after a 3 h incubation. In contrast RU38486 was extensively metabolized in cultured liver cells. The observed degration could help explain why RU38486 inhibited tyrosine aminotransferase induction in hepatoma cells at a concentration 100 times lower than that needed in liver cells. Moreover this catabolism concerned specifically the antagonist RU38486 since other steroids tested (dexamethasone, promegestone) underwent a much slower degradation. Indirect experiments suggest that the alterations of the RU38486 molecule might be at least partially related to the cytochrome P-450 which is very active in the hepatocytes. This study was paralleled by testing the effect of the antagonist on the growth of hepatoma cells. RU38486 exerted an antiproliferative effect in absence of serum. On the basis of the low metabolism of RU38486 and of its antiproliferative effect in hepatoma cells. one can emphasize that RU38486 might represent a potential drug for use in cancer therapy.