Ivan T. Oliver

Multiple forms of tyrosine aminotransferase in rat liver and their hormonal induction in the neonate

Since the discovery by Lin and Knox [l] that tyrosine aminotransferase (Gtyrosine 2-oxoglutarate aminotransferase, EC 2.6.1.5) is inducible by hydra cortisone in adult rat liver, pyridoxine [2] insulin [3] , glucagon [3,4], adrenalin and 3’,5’-cyclic AMP [5] have also been shown to increase enzyme activity in intact animals, perfused liver and liver explants in organ culture. Since the effects of some of these compounds are prevented by actinomycin D, induction of the enzyme at the genome level has been suggested. Hager and Kenney [6] and Wicks [5] have suggested multiple mechanisms for the induction of this enzyme in explanation of the diversity of inducing agents. Holt and Oliver [7,8] have reported a number of observations on tyrosine aminotransferasesynthesis in fetal and postnatal rats which are inconsistent with a unitary mechanism of induction and it was further suggested [8] that multiple forms of the enzyme with distinct and specific inductive mechanisms might better explain the apparent complexity of the system. Data presented in this paper confirm the existence of multiple forms of tyrosine aminotransferase activity and indicate differential specificity of various inducers for the various forms. Miller and Litwack [9] have also indicated the existence of at least two forms of soluble tyrosine aminotransferase in rat liver using monoiodo tyrosine and tyrosine as substrates.

Inhibitor studies on uridine diphosphoglucose pyrophosphorylase.

Abstract Uridine diphosphoglucose pyrophosphorylase from rat liver and guinea-pig brain is competitively inhibited by galactose i -phosphate. Galactose at high concentration does not affect the activity of the enzyme but inorganic orthophosphate is a non-competitive inhibitor. The possible relation of these findings to the reversible toxicity of galactose in galactosaemia is discussed.

Cytoplasmic binding of dexamethasone and induction of tyrosine aminotransferase in neonatal rat liver

Abstract Dexamethasone administration markedly increases the activity of tyrosine aminotransferase in postnatal rat liver. The glucocorticoid fails to induce the enzyme in foetal rats when administered in utero . Dexamethasone binding activity of rat liver cytoplasm is low or absent in foetal animals but increases to adult levels 1–2 days after birth. In vitro experiments with isolated nuclei indicate that foetal nuclei have the capacity to accumulate dexamethasone but only when presented with cytosol-bound glucocorticoid.

Post-Natal Changes in Blood Glucose, Phosphopyruvate Carboxylase and Tyrosine Aminotransferase after Normal Birth and Premature Delivery in the Rat

The relationship between post-natal changes in blood glucose and hepatic enzyme induction was examined in the newborn rat after delivery in the last 3 days of gestation (22 days). A period of transient hypoglycaemia followed normal birth, delivery under ether anaesthesia on day 21 and delivery without anaesthesia on day 22. When fetuses were delivered without anaesthesia on days 20 and 21 the blood glucose concentration was low at birth, was constant for 3 h then increased at a rate similar to that observed for older animals. Phosphopyruvate carboxylase and tyrosine aminotransferase developed in utero after day 21 and at term the activities were high and increased immediately after birth. The time lag for the increase in enzyme activity shortened as gestation progressed. It is concluded that postnatal changes in blood glucose concentration do not have any specific effect on hepatic enzyme induction.

Inhibition of uridine diphosphate glucose dehydrogenase by metabolic intermediates of galactose

Abstract UDPG dehydrogenase (EC 1.1.1.22) from calf liver is competitively inhibited by UDPGal in vitro and “uncompetitive” inhibition of the enzyme is shown by UDP. The K i for UDPGal is 1.33 · 10 −4 M, the K m for UDPG is 0.9–1.2 · 10 −5 M at pH 8.3. Galactose, Gal-1- P , and Glc-1- P , do not affect the enzyme activity even at high concentrations. UMP, UDP and UTP are all inhibitors. The significance of these findings is discussed in relation to a mechanism of galactose toxicity in galactosaemia and in excess galactose feeding.

The intracellular distribution of uridine diphosphate glucose starch synthetase in potato tubers.

Abstract Fractionation of subcellular particles from potato tubers in sucrose-citratte media has shown very high levels of uridine diphosphate glucose starch synthetase activity to be associated with the starch granules. No activity has been detected in other fractions. The starch granules prepared in this medium show minimal damage.

Synthesis and secretion of albumin and transferrin by foetal RAT hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.

A liver factor that interconverts multiple forms of tyrosine aminotransferase

Abstract 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.

FRACTIONATION OF RAT LIVER TYROSINE AMINOTRANSFERASE DURING THE COURSE OF PURIFICATION. FURTHER EVIDENCE FOR MULTIPLE FORMS OF THE ENZYME

Tyrosine aminotransferase (Ltyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1 S) (TAT) of rat liver has been purified by a number of workers and evidence for homogeneity has been obtained in the ultracentrifuge and by immunological criteria using Ochterlony double diffusion [l-3] . However, the electrophoresis of the purified enzyme in polyacrylamide gel has often revealed the presence of two or more bands containing enzyme activity [2-51. It has been suggested that heterogeneity detected by this method is due either to aggregation or to degradation of the protein under the conditions of eleo trophoresis. Miller and Litwack [6] have demonstrated that during the course of enzyme purification following with cortisol, the relative initial velocity of the reaction measured with monoiodotyrosine and tyrosine as substrates, shows a steady decrease. They have suggested that this phenomenon is due to formation of physical conformers of the enzyme during induction. Holt and Oliver [7] have suggested that TAT may consist of three active forms in uivo and have subsequently shown that the gel electrophoretic pattern of enzyme activity in crude liver extracts responds to different hormonal stimuli in the intact animal [8] . On the basis of these findings, the stepwise purification of the enzyme has been monitored with polyacrylamide gel electrophoresis in order

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

Abstract A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase in vitro with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated in vitro by 2.5 mM MgCl2 and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to in vitro interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of in 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.