Forms Of Tyrosine Aminotransferase Research Articles

Absence of tyrosine aminotransferase multiple forms in several mammalian animals

1. 1. Tyrosine aminotransferase multiple forms occurring in rat liver are not present in all mammalian species. 2. 2. Among animals examined only rat and mouse liver possesses multiple forms of tyrosine aminotransferase; in guinea-pig, rabbit, bovine and sheep liver the enzyme occurs in a single form. 3. 3. The presence of lysosomal converting factor (cathepsin T), responsible for arising of multiple forms of tyrosine aminotransferase in rat liver, has been checked in another species lacking enzyme subforms. Lysosomal extracts of guinea-pig liver interconverts tyrosine aminotransferase from rat liver; lysosomal extracts of rat liver does not generate multiple forms of the enzyme from guinea-pig liver. 4. 4. It has been concluded that in some animals hepatic tyrosine aminotransferase is resistant to the proteolytic cleavage by lysosomal cathepsin T.

Cathepsin T (convertase) generates the multiple forms of tyrosine aminotransferase by limited proteolysis.

Cathepsin T (convertase) generates the multiple forms of tyrosine aminotransferase by limited proteolysis.

Physical properties, limited proteolysis, and acetylation of tyrosine aminotransferase from rat liver.

The native and one of the modified forms of tyrosine aminotransferase were purified from rat liver and characterized. Several hydrodynamic properties of the native enzyme are: Stokes radius, 46 A; subunit isoelectric point, 5.6; sedimentation coefficient, 5.6 S, frictional ratio, 1.44; diffusion coefficient, 4.65 X 10(-7) cm2 s-1; extinction coefficient of a 1% solution (w:v) at 280 nm, 10.5 cm-1. The molecular weight of the dimeric protein is 110,500 as calculated from the Stokes radius and sedimentation coefficient. The subunit of the modified form is of lower molecular weight than the subunit of the native enzyme and has a pI of about 5.9. During isoelectric focusing, both forms of the enzyme separate into two components. The more acidic component that is resolved from the native enzyme is phosphorylated, but the other component is not. The amino acid composition of native tyrosine aminotransferase differs from values reported for mixtures of the three forms of this enzyme. Neither the native nor the modified forms of the enzyme possess a free alpha-amino group as judged by dansylation, nor can they be digested with leucine aminopeptidase, implying that the NH2-terminus is blocked. The possibility that tyrosine aminotransferase is acetylated was examined by translating poly(A)+RNA from hepatoma cells in a cell-free translational system in the presence and absence of inhibitors of protein acetylation. [35S]Tyrosine aminotransferase synthesized in the presence of the inhibitors has a more basic isoelectric point than the native enzyme as determined by isoelectric focusing, suggesting that the enzyme is acetylated either at the NH2-terminal or the epsilon-amino group of an internal lysine. When digested by either of two lysosomal proteases, tyrosine aminotransferase is cleaved to a smaller size. These data show that tyrosine aminotransferase is susceptible to several post-translational modifications.

Purification and characterization of a new thiol proteinase from rat kidney

The levels of activity of a new proteinase, termed cathepsin T, in rat tissues were examined. This enzyme had been previously reported to exist in rat liver on the basis of its catalysis of the conversion of multiple forms of tyrosine aminotransferase ( l-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5). Kidney was found to be the richest source of a cathepsin T Activity, exhibiting about 10-times as great a specific activity as found in liver. The order of cathepsin T activity in tissues was kidney >; >; spleen >; liver >; small intestine >; lung. The proteinase activity was not detectable in heart, skeletal muscle, brain and blood. Kidney cathepsin T was purified about 1400-fold to homogeneity with a 20% yield by the purification procedure similar to that used for the liver enzyme (Gohda, E. and Pitot, H.C. (1980) J. Biol. Chem. 255, 7371–7379). Cathepsin T purified from rat kidney was found to be a glycoprotein, the molecular weight of which was between 33 500 and 35 000. Purified kidney cathepsin T converted Form I of tyrosine aminotransferase in the same way as the liver proteinase, with concomitant conversion of 52 500 dalton subunits of the minotransferase to 48 000 dalton subunits. Kidney cathepsin T showed the same specific activities toward Form I of tyrosine aminotransferase, casein and acid-denatured hemoglobin as did the liver form of the enzyme. Many other characteristics common to the proteinases purified both from rat kidney and liver were found. We have concluded that kidney cathepsin T is the same enzyme as the liver proteinase.

Purification and characterization of a factor catalyzing the conversion of the multiple forms of tyrosine aminotransferase from rat liver.

Purification and characterization of a factor catalyzing the conversion of the multiple forms of tyrosine aminotransferase from rat liver.

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%.

Studies on the different molecular forms of tyrosine aminotransferase from rat liver and hepatoma cells

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.

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells

Corticosteroi-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 × g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I → II → III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cells subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.

Studies on the conversion of multiple forms of tyrosine aminotransferase in rat liver

Studies from several laboratories have demonstrated the existence of at least three separable forms of the hepatic enzyme, tyrosine aminotransferase. The significance of these separable forms of the enzyme isolated in vitro for the nature and regulation of the enzyme in vivo has been the subject of some controversy. The studies reported in this paper demonstrate the existence of a heat-labile, pH- and temperature-dependent, nondialyzable component associated predominantly with the lysosomal and mitochondrial fraction of rat liver which catalyzes the conversion of form II to forms III and IV of the enzyme. The activity of this conversion factor is not significantly affected by F −, molybdate ions, or two inhibitors of proteases. On the other hand, the cyanate ion completely inhibits the conversion of form II to forms III and IV of tyrosine aminotransferase, as do iodoacetate and oxidized glutathione. p-Chloromercuribenzoate also markedly inhibits the conversion. Kinetic studies suggest that the shift from one form to another follows the pathway: II to III to IV. Titration of the available sulfhydryl groups of the three forms of the enzyme demonstrates that form II possesses between 16 and 17 titratable SH groups per mole, while forms III and IV possess 15 and 13 or 14, respectively. The possible catalytic mechanism by which the conversion of the multiple forms of tyrosine aminotransferase is accomplished is discussed.

Induction of multiple forms of tyrosine aminotransferase by amino acid mixtures of different compositions

Hepatic tyrosine aminotransferase (EC 2.6.1.5) was induced in rats by intubation of amino acid mixtures (complete or tryptophan-free). Enzyme activity was increased 4-fold by the complete mixture and 8-fold by the tryptophan-free mixture. The enzyme was analyzed by chromatography on CM-Sephadex. Chromatographic patterns were characteristic of the type of inducer rather than of the chronology of the induction cycle: after induction by the complete amino acid mixture the three forms of the enzyme were equally increased whereas after induction by the tryptophan-free mixture Form I was preferentially increased.

The interconversion of multiple forms of tyrosine aminotransferase

Previous publications from this laboratory demonstrated that tyrosine aminotransferase from rat liver could be separated into three or four molecular forms by hydroxyapatite chromatography. The storage of soluble tyrosine aminotransferase for 60 hours at 4° caused a marked decrease in form II and consequent increase in form III and/or form IV while no change in total enzyme activity was seen. This effect was more marked when the liver homogenate was prepared in phosphate buffer, pH 6.9, than at pH 7.6. The pattern of multiple forms was altered by varying or changing the pH of the liver homogenate. The incubation of soluble tyrosine aminotransferase with components from the particulate fraction of a liver homogenate results in a pH dependent conversion of the multiple forms similar to that seen on storage. In addition, incubation of soluble tyrosine aminotransferase with a microsomal fraction of liver shows a similar, but less marked, effect. It is suggested, therefore, that a microsomal component whose catalytic action is dependent on pH is responsible for the interconversion of the multiple forms 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.

Problems interpreting the significance of multiple form patterns of rat liver tyrosine aminotransferase

Summary Cortisol induced tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) of rat liver cytosol can be resolved into several peaks of activity by CM-Sephadex chromatography. These separable activities have been considered distinct molecular species and are referred to as multiple forms of this enzyme. The present study demonstrates that a number of in vitro manipulations considerably alter the pattern of multiple forms of tyrosine aminotransferase. These include homogenization of the liver in buffers of varying ionic strength and pH, freezing and thawing, and sonication. Based on these findings we conclude that one must proceed cautiously when attempting to relate multiple form patterns observed in vitro to the form(s) of the enzyme in vivo .

The regulation of 4 forms of tyrosine aminotransferase in adult and developing rat liver

Tyrosine aminotransferase activity in rat liver has been separated into four isozymic forms by means of hydroxylapatite chromatography. A form (Form I) is found to be ubiquitous in tissues other than liver whereas the other three forms (II, III & IV) are present only in liver. The synthesis of form IV appears to be regulated by insulin and form II probably by corticosteroids. In fetal liver only form I is present, forms II and III appearing on the day of birth, and form IV at the time of weaning.

The role of cyclic AMP in the induction of tyrosine aminotransferase in neonatal rats: evidence for a release function based on an anomalous effect of puromycin

1. 1. In surgically delivered rat infants the administration of puromycin at 3 or 6 hours after delivery leads to potentiation of the postnatal synthesis of tyrosine aminotransferase but puromycin given at delivery effectively prevents production of enzyme activity. 2. 2. Zone sedimentation analysis shows the enzyme activity ‘ induced ’ by puromycin to be of normal size. 3. 3. Hormonal ‘ cocktails ’ containing insulin, hydrocortisone, and adrenaline produce 3 forms of tyrosine aminotransferase in 2-day postnatal rats, adrenaline produces only I form, and puromycin is an apparent inducer in some litters of animals. All these treatments produce tyrosine aminotransferase of the same sedimentation behaviour. 4. 4. Form C of the enzyme is produced by 5 hours after birth of the rat, and when compared with the multiple forms of the enzyme produced by the triple hormonal cocktail, has the same sedimentation characteristics. 5. 5. In a certain proportion of 2-day postnatal rat litters enzyme activity can be increased by adrenaline or puromycin and both agents either are consistently effective or are both ineffective. 6. 6. In ‘ inducible ’ litters, RNase digestion of either liver extracts or the polysomemicrosome fraction increases the enzyme activity in vitro but has no effect in ‘ pre-induced ’ or ‘ non-inducible ’ preparations. 7. 7. The data are interpreted as indicating the operation of a control mechanism working at an enzyme release step in the tyrosine aminotransferase system and taken together with other work it is suggested that this mechanism operates on form C of tyrosine aminotransferase and is mediated at the intracellular level by cyclic AMP.

Multiple forms of soluble rat liver tyrosine aminotransferase premature induction and postnatal development

1. 1. The postnatal development of the multiple forms of tyrosine aminotransferase in rat liver shows that form C appears within 5 hours of birth, form B appears within 10 hours of birth, and form A appears about the third postnatal day. 2. 2. Injection of triamcinolone to rat foetuses in utero results in the appearance of form C of tyrosine aminotransferase within 24 hours followed by form B. 3. 3. Treatment of foetal animals in utero with glucagon leads to the appearance of form C of the enzyme within 5 hours while the same hormone treatment of 2-day postnatal rats leads to the appearance of both forms B and C. 4. 4. In surgically delivered rats adrenalcctomized within an hour of birth, the injection of glucagon in large doses is much less effective in promoting tyrosine aminotransferase synthesis than it is in unoperated litter-mate controls or in sham-operated animals. 5. 5. The role of glucagon, adrenal steroids, adrenaline, insulin, and cyclic AMP in the postnatal induction of hepatic tyrosine aminotransferase are all discussed in terms of the patterns of multiple enzyme development.

Control of liver tyrosine aminotransferase expression: enzyme regulatory studies on inbred strains and mutant mice.

Studies on the genetic mechanisms in control of mouse liver tyrosine aminotransferase expression were of three general types: (1) studies on strain variance in endogenous enzyme activity and of various factors affecting the basal enzyme level, (2) purification of the enzyme and studies of its properties, and (3) studies of strain variance in enzyme regulation dealing primarily with glucocorticoid induction and with the starvation-induced enzyme adaptation. Tyrosine aminotransferase (l-tyrosine: 2-oxoglutarate aminotransferase, E.C. 2.6.1.5) was purified 400 to 600-fold from livers of C57BL/6J and DBA/2J inbred mice. Several of the properties of the mouse liver enzyme were similar to those known for the rat liver enzyme although the apparent Km (l-tyrosine) was lower, calculated at 6.25×10−4M. Disc gel electrophoresis of the enzyme from 105,000 g supernatant fluid after induction by hydrocortisone indicated three bands of enzyme activity with strain variance in electrophoretic mobility between the C57BL/6J and DBA/2J mice. The administration of glucose to fasting C57BL/6J mice “repressed” the starvation-induced increase in enzyme activity, but did not prevent the hydrocortisone induction of enzyme activity. DEAE-cellulose chromatography of purified enzyme from fasting DBA/2J and C57BL/6J mice which had been labeled in vivo with C 14 -l-leucine revealed strain differences in the elution patterns for both enzyme activity and radioactivity. Two peaks of enzyme activity were detected in the enzyme preparations from fasting mice. The marked strain variance in the enzyme activity and the quantity of radioactivity associated with the first enzyme peak may indicate differential rates of protein turnover for different isozymic forms of tyrosine aminotransferase. Flumethasone, a potent difluoro synthetic glucocorticoid, was used in studies on the hormonal regulation of tyrosine aminotransferase in obese mutant mice of the C57BL/6J-ob strain. The obese mice are relatively insensitive to the action of adrenal glucocorticoids to cause liver enzyme induction.

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

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

Ontogeny of soluble and mitochondrial tyrosine aminotransferases.

The development of the soluble and mitochondrial forms of tyrosine aminotransferase was observed in fetal and neonatal rhesus monkey tissues. The mitochondrial activity is detectable in early fetal life; the soluble form reaches significant activity just before the birth of the animal.